DKR polypeptides

ABSTRACT

Disclosed are nucleic acid molecules encoding novel DKR polypeptides. Also disclosed are methods of preparing the nucleic acid molecules and polypeptides, and methods of using these molecules.

This application is a continuation of U.S. Ser. No. 10/998,271, filedNov. 24, 2004, which is a continuation of U.S. Ser. No. 09/976,736,filed Oct. 9, 2001 (now abandoned), which is a divisional of U.S. Ser.No. 09/161,241, filed Sep. 25, 1998 (now U.S. Pat. No. 6,344,541), whichare hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to novel genes encoding proteins thathave use as anti-cancer therapeutics.

BACKGROUND Related Art

One of the hallmarks of cells that have become cancerous is the changein the gene expression pattern in those cells as compared to normal,non-cancerous cells. An intricate series of cell signaling events leadsto this so called “differential gene expression”, resulting inconversion of a normal cell to a cancer cell (also known as“oncogenesis” or “cell transformation”). A number of cell signalingpathways have been implicated in the process of cell transformation,such as, for example, the cadherin pathway, the delta/jagged pathway,the hedgehog/sonic hedgehog pathway, and the wnt/wingless pathway(Hunter, Cell, 88:333-346 [1997]; Currie, J. Mol. Med., 76:421-433[1998]; Peifer, Science, 275:1752-1753 [1997]. Interestingly, these samepathways are involved in cell morphogenesis, or cell differentiation,during embryo development (Hunter, supra; Cadigan et al., Genes andDevelop., 11:3286-3305 [1997]).

The wnt genes encode glycoproteins that are secreted from the cell.These glycoproteins are found in both vertebrate and invertebrateorganisms. Currently, there are at least 20 wnt family members, andthese members are believed to function variously in control of growthand in tissue differention. Recently, discovery of a novel gene wasidentified in Xenopus and mouse and has been termed dickkopf-1(“dkk-1”). This gene is purportedly a potent antagonist of wnt-8signaling (Glinka et al., Nature, 391:357-362 [1998]). Interestingly,this gene is also purportedly involved in morphogenesis in thedeveloping embryo (Glinka et al., supra). This gene thus represents anovel growth factor which may be useful in tissue regeneration, and alsorepresents a means for potentially inhibiting cell transformation viawnt signaling.

The Frzb proteins and the protein Cerberus are examples of secretedproteins that purportedly inhibit wnt signaling (Brown, Curr. OpinionCell Biol., 10:182-187 [1998).

PCT WO 98/35043, published 13 Aug. 1998 describes human SDF-5 proteinswhich are purportedly useful in regulating the binding of wntpolypeptides to their receptors.

PCT WO 98/23730, published 4 Jun. 1998, describes transfecting tumorscells with wnt-5a to purportedly decrease tumorigenicity. Wnt-5apurportedly is an antagonist of other wnts.

In view of the devastating effects of cancer, there is a need in the artto identify additional genes that may serve as antagonists of proteinsinvolved in cell transformation.

Accordingly, it is an object of this invention to provide nucleic acidmolecules and polypeptides that may be useful as anti-cancer compounds.

It is a further object to provide methods of altering the level ofexpression and/or activity of such polypeptides in the human body.

Other related objects will readily be apparent from a reading of thisdisclosure.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated nucleicacid molecule encoding a biologically active DKR polypeptide selectedfrom the group consisting of:

(a) the nucleic acid molecule comprising SEQ ID NO:1;

(b) the nucleic acid molecule comprising SEQ ID NO:2;

(c) the nucleic acid molecule comprising SEQ ID NO:3;

(d) the nucleic acid molecule comprising SEQ ID NO:4;

(e) the nucleic acid molecule comprising SEQ ID NO:5;

(f) the nucleic acid molecule comprising SEQ ID NO:6;

(g) the nucleic acid molecule comprising SEQ ID NO:7;

(h) the nucleic acid molecule comprising SEQ ID NO:75;

(i) the nucleic acid molecule comprising SEQ ID NO:76;

(j) the nucleic acid molecule comprising SEQ ID NO:77;

(k) the nucleic acid molecule comprising SEQ ID NO:78;

(l) the nucleic acid molecule encoding the polypeptide of SEQ ID NO:8;

(m) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:9;

(n) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:10, ora biologically active fragment thereof;

(o) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:11, ora biologically active fragment thereof;

(p) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:12, ora biologically active fragment thereof;

(q) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:13, ora biologically active fragment thereof;

(r) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:14, ora biologically active fragment thereof;

(s) a nucleic acid molecule that encodes a polypeptide that is at least85 percent identical to the polypeptide of SEQ ID NOs: 10, 11, 12, 13,or 14;

(t) a nucleic acid molecule that encodes a biologically active DKRpolypeptide that has 1-100 amino acid substitutions and/or deletions ascompared with the polypeptide of any of SEQ ID NOs:8, 9, 10, 11, 12, 13,or 14; and

(u) a nucleic acid molecule that hybridizes under conditions of highstringency to any of (c), (d), (e), (f), (g), (h), (i), (k), (l), (m),(n), (o), (p), (q), (r), (s), and (t) above.

In another embodiment, the invention provides an isolated nucleic acidmolecule that is the complement of any of the nucleic acid moleculesabove.

In yet another embodiment, the invention provides an isolated nucleicacid molecule encoding a biologically active DKR polypeptide selectedfrom the group of: amino acids 16-350, 21-350, 22-350, 23-350, 33-350,or 42-350, 21-145, 40-145, 40-150, 45-145, 45-145, 145-290, 150-290,300-350, or 310-350 of SEQ ID NO:9; amino acids 15-266, 24-266, or32-266 of SEQ ID NO:10; amino acids 17-259, 26-259, or 34-359 of SEQ IDNO:12; and amino acids 19-224, 20-224, 21-224, or 22-224 of SEQ IDNO:14.

In other embodiments, the invention provides vectors comprising thenucleic acid molecules, and host cells comprising the vectors.

In still another embodiment, the invention provides a process forproducing a biologically active DKR polypeptide comprising the steps of:

(a) expressing a polypeptide encoded by any of the nucleic acidmolecules herein in a suitable host; and

(b) isolating the polypeptide.

In still one other embodiment, the invention provides a biologicallyactive DKR polypeptide selected from the group consisting of:

(a) the polypeptide of SEQ ID NO:8;

(b) the polypeptide of SEQ ID NO:9;

(c) the polypeptide of SEQ ID NO:10;

(d) the polypeptide of SEQ ID NO:11;

(e) the polypeptide of SEQ ID NO:12;

(f) the polypeptide of SEQ ID NO:13;

(g) the polypeptide of SEQ ID NO:14;

(h) a polypeptide that has 1-100 amino acid substitutions or deletionsas compared with the polypeptide of any of (a)-(g) above; and

(i) a polypeptide that is at least 85 percent identical to any of thepolypeptides of (c)-(h) above.

In still one other embodiment, the invention provides the followingpolypeptides: a polypeptide that is amino acids 16-350, 21-350, 22-350,23-350, 33-350, or 42-350, 21-145, 40-145, 40-150, 45-145, 45-145,145-290, 145-300, 145-350, 150-290, 300-350, or 310-350 of FIG. 9, apolypeptide that is amino acids 15, 266, 24-266, or 32-266 of FIG. 10, apolypeptide that is amino acids 17-259, 26-259, or 34-259 of FIG. 12,and a polypeptide that is amino acids 19-224, 20-224, 21-224, or 22-224of FIG. 14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:1) depicts the cDNA sequence of mouse DKR-3.

FIG. 2 (SEQ ID NO:2) depicts the cDNA sequence of human DKR-3.

FIG. 3 (SEQ ID NO:3) depicts the DNA sequence of human DKR-1.

FIG. 4 (SEQ ID NO:4) depicts the cDNA sequence of mouse DKR-2.

FIG. 5 (SEQ ID NO:5) depicts the cDNA sequence of human DKR-2.

FIG. 6 (SEQ ID NO:6) depicts the cDNA sequence of human DKR-2a, a splicevariant of the DKR-2 gene.

FIG. 7 (SEQ ID NO:7) depicts the cDNA sequence of human DKR-4.

FIG. 8 (SEQ ID NO:8) depicts the amino acid sequence of mouse DKR-3 astranslated from the corresponding cDNA.

FIG. 9 (SEQ ID NO:9) depicts the amino acid sequence of human DKR-3 astranslated from the corresponding cDNA.

FIG. 10 (SEQ ID NO:10) depicts the amino acid sequence of human DKR-1 astranslated from the corresponding cDNA.

FIG. 11 (SEQ ID NO:11) depicts the amino acid sequence of mouse DKR-2 astranslated from the corresponding cDNA.

FIG. 12 (SEQ ID NO:12) depicts the amino acid sequence of human DKR-2 astranslated from the corresponding cDNA.

FIG. 13 (SEQ ID NO:13) depicts the amino acid sequence of human DKR-2aas translated from the corresponding cDNA.

FIG. 14 (SEQ ID NO:14) depicts the amino acid sequence of human DKR-4 astranslated from the corresponding cDNA.

FIGS. 15A-15D are photographs of Northern blots which were probed withhuman DKR-3. FIG. 15A shows the transcript level of DKR-3 in varioushuman normal (Lanes 1-2) and immortal (Lanes 3-4) cell lines, and inhuman estrogen receptor plus (“ER+”; Lanes 5-9) and estrogen receptorminus (“ER−”; Lanes 10-16) breast cancer cell lines. FIG. 15B shows thetranscript level of human DKR-3 in human normal lung cells (Lane 1), andin various human non-small cell lung cancer (“NSCLC”; Lanes 2-9) andsmall cell lung cancer (“SCLC”; Lanes 10-15) cell lines. FIG. 15C showsthe amount of transcript of human DKR-3 in five glioblastoma cell lines;three of these lines (SNB-19, U-87MG, and U-373MG) are capable offorming tumors in nude mice, while the other two lines (Hs 683 and A172) are not. FIG. 15D shows the transcript level of human DKR-3 inhuman immortal (non-cancerous) and normal cervical cells, and in humancervical cancer cell lines (indicated as “tumor cells”).

FIG. 16 is a photograph of SDS gel electrophoresis. The contents of thelanes are set forth in the Examples herein.

FIG. 17 is a photograph of SDS gel electrophoresis. The contents of thelanes are set forth in the Examples herein.

FIG. 18 is a photograph of SDS gel electrophoresis. The contents of thelanes are set forth in the Examples herein.

FIG. 19 is a photograph of SDS gel electrophoresis. The contents of thelanes are set forth in the Examples herein.

FIG. 20 is a photograph of SDS gel electrophoresis. The contents of thelanes are set forth in the Examples herein.

FIG. 21 is a photograph of a Western blot. Contents of the Lanes areindicated in the Examples herein.

FIG. 22 (SEQ ID NO:75) is a nucleic acid sequence of human DKR-1 withcodons optimized for expression in E. coli.

FIG. 23 (SEQ ID NO:76) is a nucleic acid sequence of human DKR-2 withcodons optimized for expression in E. coli.

FIG. 24 SEQ ID NO:77) is a nucleic acid sequence of human DKR-3 withcodons optimized for expression in E. coli.

FIG. 25 (SEQ ID NO:78) is a nucleic acid sequence of human DKR-4 withcodons optimized for expression in E. coli.

DETAILED DESCRIPTION OF THE INVENTION

Included in the scope of this invention are DKR polypeptides such as thepolypeptides of SEQ ID NOs:8-14, and related biologically activepolypeptide fragments, variants, and derivatives thereof.

Also included within the scope of the present invention are nucleic acidmolecules that encode DKR polypeptides such as the nucleic acidmolecules of SEQ ID Nos:1-7.

Additionally included within the scope of the present invention arenon-human mammals such as mice, rats, rabbits, goats, or sheep in whichthe gene (or genes) encoding a native DKR polypeptide has (have) beendisrupted (“knocked out”) such that the level of expression of this geneor genes is (are) significantly decreased or completely abolished. Suchmammals may be prepared using techniques and methods such as thosedescribed in U.S. Pat. No. 5,557,032. The present invention furtherincludes non-human mammals such as mice, rats, rabbits, goats, or sheepin which the gene (or genes) encoding DKR polypeptides in which eitherthe native form of the gene(s) for that mammal or a heterologous DKRpolypeptide gene(s) is (are) over expressed by the mammal, therebycreating a “transgenic” mammal. Such transgenic mammals may be preparedusing well known methods such as those described in U.S. Pat. No.5,489,743 and PCT patent application no. WO94/28122, published 8 Dec.1994. The present invention further includes non-human mammals in whichthe promoter for one or more of the DKR polypeptides of the presentinvention is either activated or inactivated (using homologousrecombination methods as described below) to alter the level ofexpression of one or more of the native DKR polypeptides.

The DKR polypeptides of the present invention are expected to haveutility as anti-cancer therapeutics for those cancers such as mammarytumors, stem cell tumors, or other cancers in which the wnt and/or sonichedgehog (shh) signal transduction pathways are activated. Specific wntmembers can transform mammary tissue (Hunter, supra) and are abnormallyexpressed in many human tumors (Huguet, Cancer Res., 54:2615-2621[1994]; Dale, Cancer Res., 56:4320-4323 [1996]; see also PCT WO97/39357). Such activity is expected in view of data presented herein inwhich the level of DKR-3 transcript is decreased or not detectable atall in many cancer cell lines as compared to similar normal cell lines.Further, such activity is expected in view of the relationship of thegenes and polypeptides of the present invention to the gene dickkopf-1(which, as mentioned above, is purportedly a potent antagonist ofwnt-8). DKR-1, a novel gene of the present invention, is a humanortholog of dkk-1. DKR-2, DKR-3, and DKR-4, all novel genes of thepresent invention, are each related to DKR-1 by their cysteine pattern.In particular, these DKR polypeptides may be of use for treatment ofstem cell tumors, mammary tumors, and other cancers in which wnt genesare expressed, and in cancers where wnt and/or shh signaling isactivated.

The DKR polypeptides of the present invention may also be administeredas agents that can induce and/or enhance tissue differentiation, such asbone formation, cartilage formation, muscle tissue formation, nervetissue formation, and hematopoietic cell formation. Such activities areexpected in view of the fact that a) Xenopus dkk-1 purportedly promoteshead induction, heart formation, and differentiation or the developingCNS (Glinka, supra); and b) certain wnt polypeptides appear to functionin embryo development (Cadigan, Genes and Devel., 11:3286-3305 [1997]),specifically development of the pituitary (Treier, Genes and Devel.,12:1691-1704 [1998]), myogenesis (Munsterberg et al., Genes and Devel.,9:2911-2922 [1995]), osteogenesis (PCT WO 95/17416; PCT WO98/16641),kidney development (Stark et al., Nature 372:679-683 [1994]),development of the CNS (Dickinson et al., Development, 120:1453-1471[1994]), and hematopoiesis (PCT WO 98/06747). Thus, addition of certainDKR polypeptides in such cell cultures or tissues may serve to modifythe activity of various wnt polypeptides in cellular differentiationprocesses.

The DKR polypeptides herein may be used in either an in vivo manner oran ex vivo manner for such applications. For example, one or more of theDKR polypeptides of the present invention may be added to a culture ofcartilage tissue or nerve tissue, or hematopoietic stem cells, eitheralone, or in combination with other growth factors and/or other tissuedifferentiation factors, so as to induce or enhance the regeneration ofsuch tissues. Alternatively, such DKR polypeptides of the presentinvention may, for example, be injected directly into a joint in need ofcartilage, into the spinal cord where the cord has been damaged, intodamaged brain tissue, or into bone marrow to enhance hematopoiesis.

The term “DKR polypeptides” as used herein refers to any protein orpolypeptide having the properties described herein for DKR polypeptides.The DKR polypeptides may or may not have amino terminal methionines,depending on the manner in which they are prepared. By way ofillustration, DKR polypeptides refers to (1) a biologically activepolypeptide encoded by any of the DKR polypeptides nucleic acidmolecules as defined in any of items (a)-(f) below; (2) naturallyoccurring allelic variants and synthetic variants of any of DKRpolypeptide in which one or more amino acid substitutions, deletions,and/or insertions are present as compared to the DKR polypeptides of SEQID NOs:8-14, and/or (3) biologically active polypeptides, or fragmentsor variants thereof, that have been chemically modified.

As used herein, the term “DKR polypeptide fragment” refers to a peptideor polypeptide that is less than the full length amino acid sequence ofa naturally occurring DKR polypeptide but has the biological activity ofany of the DKR polypeptides provided herein. Such a fragment may betruncated at the amino terminus, the carboxy terminus, and/or internally(such as by natural splicing), and may be a variant or a derivative ofany of the DKR polypeptides. Such DKR polypeptides fragments may beprepared with or without an amino terminal methionine. In addition, DKRpolypeptides fragments can be naturally occurring fragments such as DKRpolypeptide splice variants (SEQ ID NO:13), other splice variants, andfragments resulting from naturally occurring in vivo protease activity.Preferred DKR polypeptide fragments include amino acids 16-350, 21-350,22-350, 23-350, 33-350, 42-350, 21-145, 40-145, 40-150, 45-145, 145-290,145-300, 145-350, 150-290, 300-350, and 310-350, of SEQ ID NO:9; aminoacids 15-266, 24-266, or 32-266 of SEQ ID NO:10; amino acids 17-259,26-259, or 34-359 of SEQ ID NO:12; and amino acids 19-224, 20-224,21-224, or 22-224 of SEQ ID NO:14.

As used herein, the term “DKR polypeptide variants” refers to DKRpolypeptides whose amino acid sequences contain one or more amino acidsequence substitutions, deletions, and/or insertions as compared to theDKR polypeptides amino acid sequences set forth in SEQ ID NOS:8-14. SuchDKR polypeptides variants can be prepared from the corresponding DKRpolypeptides nucleic acid molecule variants, which have a DNA sequencethat varies accordingly from the DNA sequences for wild type DKRpolypeptides as set forth in SEQ ID NOS:7-14. Preferred variants of thehuman DKR polypeptides include alanine substitutions at one or more ofamino acid positions. Other preferred substitutions include conservativesubstitutions at the amino acid positions indicated in the Examplesherein, as well as those encoded by DKR nucleic acid molecules asdescribed below.

As used herein, the term “DKR polypeptide derivatives” refers to DKRpolypeptides, variants, or fragments thereof, that have been chemicallymodified, as for example, by addition of one or more polyethylene glycolmolecules, sugars, phosphates, and/or other such molecules, where themolecule or molecules are not naturally attached to wild-type DKRpolypeptides.

As used herein, the terms “biologically active DKR polypeptides”,“biologically active DKR polypeptide fragments”, “biologically activeDKR polypeptide variants”, and “biologically active DKR polypeptidederivatives” refer to DKR polypeptides that have the ability to decreasecancer cell proliferation in the Anchorage Independent Growth Assay ofExample 12 herein, or in the In Vivo Tumor Assay of Example 13 herein,or in both assays.

As used herein, the term “DKR polypeptide nucleic acids” when used todescribe a nucleic acid molecule refers to a nucleic acid molecule orfragment thereof that (a) has the nucleotide sequence as set forth inany of SEQ ID NOs:1-7; (b) has a nucleic acid sequence encoding apolypeptide that is at least 85 percent identical, but may be greaterthan 85 percent, i.e., 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent identical to the polypeptide encoded by any of SEQ IDNOS:10-14; (c) is a naturally occurring allelic variant or alternatesplice variant of (a) or (b); (d) is a nucleic acid variant of (a)-(c)produced as provided for herein; (e) has a sequence that iscomplementary to (a)-(d); (f) hybridizes to any of (a)-(e) underconditions of high stringency and/or (g) has a nucleic acid sequenceencoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, orup to 100 amino acid substitutions and/or deletions of any mature DKRpolypeptide (i.e., a DKR polypeptide with its endogenous signal peptideremoved).

Percent sequence identity can be determined by standard methods that arecommonly used to compare the similarity in position of the amino acidsof two polypeptides. By way of example, using a computer algorithm suchas GAP (Genetic Computer Group, University of Wisconsin, Madison, Wis.),the two polypeptides for which the percent sequence identity is to bedetermined are aligned for optimal matching of their respective aminoacids (the “matched span”, as determined by the algorithm). A gapopening penalty (which is calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid by the particular comparison matrix) and a gapextension penalty (which is usually 1/10 times the gap opening penalty),as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used inconjunction with the algorithm. A standard comparison matrix (seeDayhoff et al., in: Atlas of Protein Sequence and Structure, vol. 5,supp. 3 [1978] for the PAM250 comparison matrix; see Henikoff et al.,Proc. Natl. Acad. Sci. USA, 89:10915-10919 [1992] for the BLOSUM 62comparison matrix) is also used by the algorithm. The percent identityis then calculated by the algorithm by determining the percent identityas follows:

$\frac{\begin{matrix}{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}} \\{{in}\mspace{14mu}{the}\mspace{14mu}{matched}\mspace{14mu}{span}}\end{matrix}}{\begin{matrix}\left\lbrack {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}} \right. \\\begin{matrix}{\left. {{within}\mspace{14mu}{the}\mspace{14mu}{matched}\mspace{14mu}{span}} \right\rbrack +} \\\begin{matrix}\left\lbrack {{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}} \right. \\\begin{matrix}{{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{20mu}{to}} \\\left. {{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}} \right\rbrack\end{matrix}\end{matrix}\end{matrix}\end{matrix}} \times 100$Polypeptides that are at least 85 percent identical will typically haveone or more amino acid substitutions, deletions, and/or insertions ascompared with any of the wild type DKR polypeptides. Usually, thesubstitutions of the native residue will be either alanine, or aconservative amino acid so as to have little or no effect on the overallnet charge, polarity, or hydrophobicity of the protein. Conservativesubstitutions are set forth in Table I below.

TABLE I Conservative Amino Acid Substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Uncharged Polar: glutamineasparagine serine threonine tyrosine Non-Polar: phenylalanine tryptophancysteine glycine alanine valine proline methionine leucine isoleucine

The term “conditions of high stringency” refers to hybridization andwashing under conditions that permit binding of a nucleic acid moleculeused for screening, such as an oligonucleotide probe or cDNA moleculeprobe, to highly homologous sequences. An exemplary high stringency washsolution is 0.2×SSC and 0.1 percent SDS used at a temperature of between50° C.-65° C.

Where oligonucleotide probes are used to screen cDNA or genomiclibraries, one of the following two high stringency solution may beused. The first of these is 6×SSC with 0.05 percent sodium pyrophosphateat a temperature of 35° C.-62° C., depending on the length of theoligonucleotide probe. For example, 14 base pair probes are washed at35-40° C., 17 base pair probes are washed at 45-50° C., 20 base pairprobes are washed at 52-57° C., and 23 base pair probes are washed at57-63° C. The temperature can be increased 2-3° C. where the backgroundnon-specific binding appears high. A second high stringency solutionutilizes tetramethylammonium chloride (TMAC) for washing oligonucleotideprobes. One stringent washing solution is 3 M TMAC, 50 mM Tris-HCl, pH8.0, and 0.2 percent SDS. The washing temperature using this solution isa function of the length of the probe. For example, a 17 base pair probeis washed at about 45-50° C.

As used herein, the terms “effective amount” and “therapeuticallyeffective amount” refer to the amount of a DKR polypeptide necessary tosupport one or more biological activities of the DKR polypeptides as setforth above.

A full-length DKR polypeptide or fragment thereof can be prepared usingwell known recombinant DNA technology methods such as those set forth inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]) and/or Ausubelet al., eds., (Current Protocols in Molecular Biology, Green PublishersInc. and Wiley and Sons, N.Y. [1994]). A gene or cDNA encoding a DKRpolypeptide or fragment thereof may be obtained for example by screeninga genomic or cDNA library, or by PCR amplification. Probes or primersuseful for screening the library can be generated based on sequenceinformation for other known genes or gene fragments from the same or arelated family of genes, such as, for example, conserved motifs found inother DKR polypeptides such as the cysteine pattern. In addition, wherea gene encoding DKR polypeptide has been identified from one species,all or a portion of that gene may be used as a probe to identifyhomologous genes from other species. The probes or primers may be usedto screen cDNA libraries from various tissue sources believed to expressthe DKR gene. Typically, conditions of high stringency will be employedfor screening to minimize the number of false positives obtained fromthe screen.

Another means to prepare a gene encoding a DKR polypeptide or fragmentthereof is to employ chemical synthesis using methods well known to theskilled artisan such as those described by Engels et al. (Angew. Chem.Intl. Ed., 28:716-734 [1989]). These methods include, inter alia, thephosphotriester, phosphoramidite, and H-phosphonate methods for nucleicacid synthesis. A preferred method for such chemical synthesis ispolymer-supported synthesis using standard phosphoramidite chemistry.Typically, the DNA encoding the DKR polypeptide will be several hundrednucleotides in length. Nucleic acids larger than about 100 nucleotidescan be synthesized as several fragments using these methods. Thefragments can then be ligated together to form the full length DKRpolypeptide. Usually, the DNA fragment encoding the amino terminus ofthe polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of the DKRpolypeptide, depending on whether the polypeptide produced in the hostcell is designed to be secreted from that cell.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of the naturally occurring DKR polypeptides. Nucleic acidvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well. Preferred nucleic acid variants are thosecontaining nucleotide substitutions accounting for codon preference inthe host cell that is to be used to produce the DKR polypeptide(s). Such“codon optimization” can be determined via computer algorithers whichincorporate codon frequency tables such as “Ecohigh. Cod” for codonpreference of highly expressed bacterial genes as provided by theUniversity of Wisconsin Package Version 9.0, Genetics Computer Group,Madison, Wis. Other useful codon frequency tables include“Celegans_high.cod”, “Celegans_low.cod”, “Drosophila_high.cod”,“Human_high.cod”, “Maize_high.cod”, and “Yeast_high.cod”. Otherpreferred variants are those encoding conservative amino acid changes asdescribed above (e.g., wherein the charge or polarity of the naturallyoccurring amino acid side chain is not altered substantially bysubstitution with a different amino acid) as compared to wild type,and/or those designed to either generate a novel glycosylation and/orphosphorylation site(s), or those designed to delete an existingglycosylation and/or phosphorylation site(s).

The gene, cDNA, or fragment thereof encoding the DKR polypeptide can beinserted into an appropriate expression or amplification vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). The gene, cDNA orfragment thereof encoding the DKR polypeptide may be amplified/expressedin prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotichost cells. Selection of the host cell will depend in part on whetherthe DKR polypeptide or fragment thereof is to be glycosylated and/orphosphorylated. If so, yeast, insect, or mammalian host cells arepreferable.

Typically, the vectors used in any of the host cells will contain 5′flanking sequence (also referred to as a “promoter”) and otherregulatory elements as well such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a completeintron sequence containing a donor and acceptor splice site, a signalpeptide sequence, a ribosome binding site element, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these elements is discussed below. Optionally, the vector may containa “tag” sequence, i.e., an oligonucleotide molecule located at the 5′ or3′ end of the DKR polypeptide coding sequence; the oligonucleotidemolecule encodes polyHis (such as hexaHis), or other “tag” such as FLAG,HA (hemaglutinin Influenza virus) or myc for which commerciallyavailable antibodies exist. This tag is typically fused to thepolypeptide upon expression of the polypeptide, and can serve as meansfor affinity purification of the DKR polypeptide from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purified DKRpolypeptide by various means such as using certain peptidases.

The human immunoglobulin hinge and Fc region could be fused at eitherthe N-terminus or C-terminus of the DKR polypeptides by one skilled inthe art. The subsequent Fc-fusion protein could be purified by use of aProtein A affinity column. Fc is known to exhibit a long pharmacokinetichalf-life in vivo and proteins fused to Fc have been found to exhibit asubstantially greater half-life in vivo than the unfused counterpart.Also, fusion to the Fc region allows for dimerization/multimerization ofthe molecule that may be useful for the bioactivity of some molecules.

The 5′ flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5′ flanking sequences from more than one source), synthetic, or itmay be the native DKR polypeptides gene 5′ flanking sequence. As such,the source of the 5′ flanking sequence may be any unicellularprokaryotic or eukaryotic organism, any vertebrate or invertebrateorganism, or any plant, provided that the 5′ flanking sequence isfunctional in, and can be activated by, the host cell machinery.

The 5′ flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically, 5′flanking sequences useful herein other than the DKR gene flankingsequence will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of the 5′ flanking sequence maybe known. Here, the 5′ flanking sequence may be synthesized using themethods described above for nucleic acid synthesis or cloning.

Where all or only a portion of the 5′ flanking sequence is known, it maybe obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or 5′ flanking sequence fragments from thesame or another species.

Where the 5′ flanking sequence is not known, a fragment of DNAcontaining a 5′ flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, Qiagen® column or other methodsknown to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe DKR polypeptide. If the vector of choice does not contain an originof replication site, one may be chemically synthesized based on a knownsequence, and ligated into the vector.

The transcription termination element is typically located 3′ of the endof the DKR polypeptide coding sequence and serves to terminatetranscription of the DKR polypeptide. Usually, the transcriptiontermination element in prokaryotic cells is a G-C rich fragment followedby a poly T sequence. While the element is easily cloned from a libraryor even purchased commercially as part of a vector, it can also bereadily synthesized using methods for nucleic acid synthesis such asthose described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translation initiation of mRNA. The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of the DKRpolypeptide to be synthesized. The Shine-Dalgarno sequence is varied butis typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for DKR polypeptide to be secretedfrom the host cell, a signal sequence may be used to direct the DKRpolypeptide out of the host cell where it is synthesized, and thecarboxy-terminal part of the protein may be deleted in order to preventmembrane anchoring. Typically, the signal sequence is positioned in thecoding region of the DKR gene or cDNA, or directly at the 5′ end of theDKR gene coding region. Many signal sequences have been identified, andany of them that are functional in the selected host cell may be used inconjunction with the DKR gene or cDNA. Therefore, the signal sequencemay be homologous or heterologous to the DKR gene or cDNA, and may behomologous or heterologous to the DKR polypeptides gene or cDNA.Additionally, the signal sequence may be chemically synthesized usingmethods set forth above.

In most cases, secretion of the polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the aminoterminal methionine from the polypeptide.

In many cases, transcription of the DKR gene or cDNA is increased by thepresence of one or more introns in the vector; this is particularly truewhere the DKR polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the DKR gene, especially where the gene used is a fulllength genomic sequence or a fragment thereof. Where the intron is notnaturally occurring within the gene (as for most cDNAs), the intron(s)may be obtained from another source. The position of the intron withrespect to the 5′ flanking sequence and the DKR gene is generallyimportant, as the intron must be transcribed to be effective. As such,where the DKR gene inserted into the expression vector is a cDNAmolecule, the preferred position for the intron is 3′ to thetranscription start site, and 5′ to the polyA transcription terminationsequence. Preferably for DKR cDNA, the intron or introns will be locatedon one side or the other (i.e., 5′ or 3′) of the cDNA such that it doesnot interrupt the this coding sequence. Any intron from any source,including any viral, prokaryotic and eukaryotic (plant or animal)organisms, may be used to practice this invention, provided that it iscompatible with the host cell(s) into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this invention are typicallyconstructed from a starting vectors such as a commercially availablevector. Such vectors may or may not contain some of the elements to beincluded in the completed vector. If none of the desired elements arepresent in the starting vector, each element may be individually ligatedinto the vector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to “blunt” the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in “sticky ends” using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra.

Alternatively, two or more of the elements to be inserted into thevector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

One other method for constructing the vector to conduct all ligations ofthe various elements simultaneously in one reaction mixture. Here, manynonsense or nonfunctional vectors will be generated due to improperligation or insertion of the elements, however the functional vector maybe identified and selected by restriction endonuclease digestion.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15b (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding full length or truncated DKR polypeptide has been inserted intothe proper site of the vector, the completed vector may be inserted intoa suitable host cell for amplification and/or polypeptide expression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, cansynthesize DKR polypeptide which can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Aftercollection, the DKR polypeptide can be purified using methods such asmolecular sieve chromatography, affinity chromatography, and the like.

Selection of the host cell for DKR polypeptide production will depend inpart on whether the DKR polypeptide is to be glycosylated orphosphorylated (in which case eukaryotic host cells are preferred), andthe manner in which the host cell is able to “fold” the protein into itsnative tertiary structure (e.g., proper orientation of disulfidebridges, etc.) such that biologically active protein is prepared by theDKR polypeptide that has biological activity, the DKR polypeptide may be“folded” after synthesis using appropriate chemical conditions asdiscussed below.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO), human embryonic kidney (HEK) 293 or 293Tcells, or 3T3 cells. The selection of suitable mammalian host cells andmethods for transformation, culture, amplification, screening andproduct production and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 and COS-7 cell lines, and theCV-1 cell line. Further exemplary mammalian host cells include primatecell lines and rodent cell lines, including transformed cell lines.Normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, as well as primary explants, are also suitable.Candidate cells may be genotypically deficient in the selection gene, ormay contain a dominantly acting selection gene. Other suitable mammaliancell lines include but are not limited to, mouse neuroblastoma N2Acells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c orNIH mice, BHK or HaK hamster cell lines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniques, 14:810-817 [1993]), Lucklow (Curr. Opin.Biotechnol., 4:564-572 [1993]) and Lucklow et al. (J. Virol.,67:4566-4579 [1993]). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

Insertion (also referred to as “transformation” or “transfection”) ofthe vector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The media will usually contain all nutrients necessary for the growthand survival of the cells. Suitable media for culturing E. coli cellsare for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitablemedia for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all ofwhich may be supplemented with serum and/or growth factors as requiredby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of DKR polypeptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If the DKR polypeptide has been designed to be secreted from the hostcells, the majority of polypeptide may be found in the cell culturemedium. Polypeptides prepared in this way will typically not possess anamino terminal methionine, as it is removed during secretion from thecell. If however, the DKR polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram negative bacteriahost cells) and may have an amino terminal methionine.

For DKR polypeptide situated in the host cell cytoplasm and/or nucleus,the host cells are typically first disrupted mechanically or withdetergent to release the intra-cellular contents into a bufferedsolution. DKR polypeptide can then be isolated from this solution.

Purification of DKR polypeptide from solution can be accomplished usinga variety of techniques. If the polypeptide has been synthesized suchthat it contains a tag such as Hexahistidine (DKR polypeptide/hexaHis)or other small peptide such as FLAG (Eastman Kodak Co., New Haven,Conn.) or myc (Invitrogen, Carlsbad, Calif.) at either its carboxyl oramino terminus, it may essentially be purified in a one-step process bypassing the solution through an affinity column where the column matrixhas a high affinity for the tag or for the polypeptide directly (i.e., amonoclonal antibody specifically recognizing DKR polypeptide). Forexample, polyhistidine binds with great affinity and specificity tonickel, thus an affinity column of nickel (such as the Qiagen® nickelcolumns) can be used for purification of DKR polypeptide/polyHis. (Seefor example, Ausubel et al., eds., Current Protocols in MolecularBiology, Section 10.11.8, John Wiley & Sons, New York [1993]).

Where the DKR polypeptide is prepared without a tag attached, and noantibodies are available, other well known procedures for purificationcan be used. Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific).In some cases, two or more of these techniques may be combined toachieve increased purity.

If it is anticipated that the DKR polypeptide will be found primarilyintracellularly, the intracellular material (including inclusion bodiesfor gram-negative bacteria) can be extracted from the host cell usingany standard technique known to the skilled artisan. For example, thehost cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

If the DKR polypeptide has formed inclusion bodies in the cytosol, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The DKR polypeptide in its now soluble form can thenbe analyzed using gel electrophoresis, immunoprecipitation or the like.If it is desired to isolate the DKR polypeptide, isolation may beaccomplished using standard methods such as those set forth below and inMarston et al. (Meth. Enz., 182:264-275 [1990]). In some cases, the DKRpolypeptide may not be biologically active upon isolation. Variousmethods for “refolding” or converting the polypeptide to its tertiarystructure and generating disulfide linkages, can be used to restorebiological activity. Such methods include exposing the solubilizedpolypeptide to a pH usually above 7 and in the presence of a particularconcentration of a chaotrope. The selection of chaotrope is very similarto the choices used for inclusion body solubilization but usually at alower concentration and is not necessarily the same chaotrope as usedfor the solubilization. In most cases the refolding/oxidation solutionwill also contain a reducing agent or the reducing agent plus its'oxidized form in a specific ratio to generate a particular redoxpotential allowing for disulfide shuffling to occur in the formation ofthe protein's cysteine bridge(s). Some of the commonly used redoxcouples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH,cupric chloride, dithiothreitol (DTT)/dithiane DTT, 2-mercaptoethanol(bME)/dithio-b (ME). In many instances a cosolvent is necessary toincrease the efficiency of the refolding and the more common reagentsused for this purpose include glycerol, polyethylene glycol of variousmolecular weights, and arginine.

If DKR polypeptide inclusion bodies are not formed to a significantdegree in the host cell, the DKR polypeptide will be found primarily inthe supernatant after centrifugation of the cell homogenate, and the DKRpolypeptide can be isolated from the supernatant using methods such asthose set forth below.

In those situations where it is preferable to partially or completelyisolate the DKR polypeptide, purification can be accomplished usingstandard methods well known to the skilled artisan. Such methodsinclude, without limitation, separation by electrophoresis followed byelectroelution, various types of chromatography (immunoaffinity,molecular sieve, and/or ion exchange), and/or high pressure liquidchromatography. In some cases, it may be preferable to use more than oneof these methods for complete purification.

In addition to preparing and purifying DKR polypeptide using recombinantDNA techniques, the DKR polypeptides, fragments, and/or derivativesthereof may be prepared by chemical synthesis methods (such as solidphase peptide synthesis) using techniques known in the art such as thoseset forth by Merrifield et al., (J. Am. Chem. Soc., 85:2149 [1963]),Houghten et al. (Proc Natl Acad. Sci. USA, 82:5132 [1985]), and Stewartand Young (Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford,Ill. [1984]). Such polypeptides may be synthesized with or without amethionine on the amino terminus. Chemically synthesized DKRpolypeptides or fragments may be oxidized using methods set forth inthese references to form disulfide bridges. The DKR polypeptides orfragments are expected to have biological activity comparable to DKRpolypeptides produced recombinantly or purified from natural sources,and thus may be used interchangeably with recombinant or natural DKRpolypeptide.

Chemically modified DKR polypeptide compositions in which DKRpolypeptide is linked to a polymer are included within the scope of thepresent invention. The polymer selected is typically water soluble sothat the protein to which it is attached does not precipitate in anaqueous environment, such as a physiological environment. The polymerselected is usually modified to have a single reactive group, such as anactive ester for acylation or an aldehyde for alkylation, so that thedegree of polymerization may be controlled as provided for in thepresent methods. The polymer may be of any molecular weight, and may bebranched or unbranched. Included within the scope of DKR polypeptidepolymers is a mixture of polymers. Preferably, for therapeutic use ofthe end-product preparation, the polymer will be pharmaceuticallyacceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol.

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A preferredreactive aldehyde is polyethylene glycol propionaldehyde, which is waterstable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S.Pat. No. 5,252,714).

Pegylation of DKR polypeptides may be carried out by any of thepegylation reactions known in the art, as described for example in thefollowing references: Focus on Growth Factors 3: 4-10 (1992); EP 0 154316; and EP 0 401 384. Preferably, the pegylation is carried out via anacylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer) as described below.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated DKRpolypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby DKR polypeptidebecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG:protein, thegreater the percentage of poly-pegylated product.

Generally, conditions which may be alleviated or modulated byadministration of the present polymer/polypeptides include thosedescribed herein for DKR polypeptides molecules. However, thepolymer/DKR polypeptides molecules disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

The DKR polypeptides, fragments thereof, variants, and derivatives, maybe employed alone, together, or in combination with other pharmaceuticalcompositions. The DKR polypeptides, fragments, variants, and derivativesmay be used in combination with cytokines, growth factors, antibiotics,anti-inflammatories, and/or chemotherapeutic agents as is appropriatefor the indication being treated.

DKR nucleic acid molecules, fragments, and/or derivatives that do notthemselves encode polypeptides that are active in activity assays may beuseful as hybridization probes in diagnostic assays to test, eitherqualitatively or quantitatively, for the presence of DKR DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

DKR polypeptide fragments, variants, and/or derivatives that are notthemselves active in activity assays may be useful for preparingantibodies that recognize DKR polypeptides.

The DKR polypeptides, fragments, variants, and/or derivatives may beused to prepare antibodies using standard methods. Thus, antibodies thatreact with the DKR polypeptides, as well as reactive fragments of suchantibodies, are also contemplated as within the scope of the presentinvention. The antibodies may be polyclonal, monoclonal, recombinant,chimeric, single-chain and/or bispecific. Typically, the antibody orfragment thereof will either be of human origin, or will be “humanized”,i.e., prepared so as to prevent or minimize an immune reaction to theantibody when administered to a patient. The antibody fragment may beany fragment that is reactive with DKR polypeptides of the presentinvention, such as, F_(ab), F_(ab)′, etc. Also provided by thisinvention are the hybridomas generated by presenting any DKR polypeptideor fragments thereof as an antigen to a selected mammal, followed byfusing cells (e.g., spleen cells) of the mammal with certain cancercells to create immortalized cell lines by known techniques. The methodsemployed to generate such cell lines and antibodies directed against allor portions of a human DKR polypeptide of the present invention are alsoencompassed by this invention.

The antibodies may be used therapeutically, such as to inhibit bindingof the DKR polypeptide to its binding partner. The antibodies mayfurther be used for in vivo and in vitro diagnostic purposes, such as inlabeled form to detect the presence of DKR polypeptide in a body fluidor cell sample.

Preferred antibodies are human antibodies, either polyclonal ormonoclonal.

Therapeutic Compositions and Administration

Therapeutic compositions of DKR polypeptides are within the scope of thepresent invention. Such compositions may comprise a therapeuticallyeffective amount of the polypeptide or fragments, variants, orderivatives in admixture with a pharmaceutically acceptable carrier. Thecarrier material may be water for injection, preferably supplementedwith other materials common in solutions for administration to mammals.Typically, a DKR polypeptide therapeutic compound will be administeredin the form of a composition comprising purified polypeptide, fragment,variant, or derivative in conjunction with one or more physiologicallyacceptable carriers, excipients, or diluents. Neutral buffered saline orsaline mixed with serum albumin are exemplary appropriate carriers.Preferably, the product is formulated as a lyophilizate usingappropriate excipients (e.g., sucrose). Other standard carriers,diluents, and excipients may be included as desired. Other exemplarycompositions comprise Tris buffer of about pH 7.0-8.5, or acetate bufferof about pH 4.0-5.5, which may further include sorbitol or a suitablesubstitute therefor.

The DKR polypeptide compositions can be administered parenterally.Alternatively, the compositions may be administered intravenously orsubcutaneously. When systemically administered, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, with due regardto pH, isotonicity, stability and the like, is within the skill of theart.

Therapeutic formulations of DKR polypeptide compositions useful forpracticing the present invention may be prepared for storage by mixingthe selected composition having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company [1990]) in the form of a lyophilized cake or anaqueous solution. Acceptable carriers, excipients or stabilizers arenontoxic to recipients and are preferably inert at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,or other organic acids; antioxidants such as ascorbic acid; lowmolecular weight polypeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, pluronics orpolyethylene glycol (PEG).

An effective amount of the DKR polypeptide composition(s) to be employedtherapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which the DKR polypeptide is beingused, the route of administration, and the condition of the patient.Accordingly, it will be necessary for the therapist to titer the dosageand modify the route of administration as required to obtain the optimaltherapeutic effect. A typical daily dosage may range from about 0.1μg/kg to up to 100 mg/kg or more, depending on the factors mentionedabove. Typically, a clinician will administer the composition until adosage is reached that achieves the desired effect. The composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of DKR polypeptide) overtime, or as a continuous infusion via implantation device or catheter.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, the type of disorder under treatment, the age and generalhealth of the recipient, will be able to ascertain proper dosing.

The DKR polypeptide composition to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes. Where the composition is lyophilized,sterilization using these methods may be conducted either prior to, orfollowing, lyophilization and reconstitution. The composition forparenteral administration ordinarily will be stored in lyophilized formor in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of administration of the composition is in accord with knownmethods, e.g. oral, injection or infusion by intravenous,intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems or implantationdevice which may optionally involve the use of a catheter. Wheredesired, the compositions may be administered continuously by infusion,bolus injection or by implantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which DKR polypeptide has beenabsorbed.

Where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of DKR polypeptide may bedirectly through the device via bolus, or via continuous administration,or via catheter using continuous infusion.

DKR polypeptide may be administered in a sustained release formulationor preparation. Suitable examples of sustained-release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al, Biopolymers, 22: 547-556 [1983]),poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982]),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also may include liposomes, which can be prepared by any ofseveral methods known in the art (e.g., Eppstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 [1985]; EP 36,676; EP 88,046; EP 143,949).

In some cases, it may be desirable to use DKR polypeptide compositionsin an ex vivo manner. Here, cells, tissues, or organs that have beenremoved from the patient are exposed to DKR polypeptide compositionsafter which the cells, tissues and/or organs are subsequently implantedback into the patient.

In other cases, DKR polypeptide may be delivered through implanting intopatients certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides, fragments, variants, or derivatives. Such cells may beanimal or human cells, and may be derived from the patient's own tissueor from another source, either human or non-human. Optionally, the cellsmay be immortalized. However, in order to decrease the chance of animmunological response, it is preferred that the cells be encapsulatedto avoid infiltration of surrounding tissues. The encapsulationmaterials are typically biocompatible, semi-permeable polymericenclosures or membranes that allow release of the protein product(s) butprevent destruction of the cells by the patient's immune system or byother detrimental factors from the surrounding tissues.

Methods used for membrane encapsulation of cells are familiar to theskilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627. A system for encapsulating living cells is described in PCTWO 91/10425 (Aebischer et al.). Techniques for formulating a variety ofother sustained or controlled delivery means, such as liposome carriers,bio-erodible particles or beads, are also known to those in the art, andare described, for example, in U.S. Pat. No. 5,653,975 (Baetge et al.,CytoTherapeutics, Inc.). The cells, with or without encapsulation, maybe implanted into suitable body tissues or organs of the patient.

As discussed above, it may be desirable to treat isolated cellpopulations such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like with one or more DKR polypeptides,variants, derivatives and/or fragments. This can be accomplished byexposing the isolated cells to the polypeptide, variant, derivative, orfragment directly, where it is in a form that is permeable to the cellmembrane. Alternatively, gene therapy can be employed as describedbelow.

One manner in which gene therapy can be applied is to use the DKR gene(either genomic DNA, cDNA, and/or synthetic DNA encoding a DKRpolypeptide, or a fragment, variant, or derivative thereof) which may beoperably linked to a constitutive or inducible promoter to form a “genetherapy DNA construct”. The promoter may be homologous or heterologousto the endogenous DKR gene, provided that it is active in the cell ortissue type into which the construct will be inserted. Other componentsof the gene therapy DNA construct may optionally include, as required,DNA molecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (as, for example, for cell targeting)cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as factors to enable vectormanufacture.

This gene therapy DNA construct can then be introduced into thepatient's cells (either ex vivo or in vivo). One means for introducingthe gene therapy DNA construct is via viral vectors. Suitable viralvectors typically used in gene therapy for delivery of gene therapy DNAconstructs include, without limitation, adenovirus, adeno-associatedvirus, herpes simplex virus, lentivirus, papilloma virus, and retrovirusvectors. Some of these vectors, such as retroviral vectors, will deliverthe gene therapy DNA construct to the chromosomal DNA of the patient'scells, and the gene therapy DNA construct can integrate into thechromosomal DNA; other vectors will function as episomes and the genetherapy DNA construct will remain in the cytoplasm. The use of genetherapy vectors is described, for example, in U.S. Pat. Nos. 5,672,344(30 Sep. 1997; Kelly et al., University of Michigan), 5,399,346 (21 Mar.1995; Anderson et al., U.S Dept. Health and Human Services), 5,631,236(20 May 1997; Woo et al., Baylor College of Medicine), and 5,635,399 (3Jun. 1997; Kriegler et al., Chiron Corp.).

Alternative means to deliver gene therapy DNA constructs to a patient'scells without the use of viral vectors include, without limitation,liposome-mediated transfer, direct injection of naked DNA,receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,“gene gun”). See U.S. Pat. Nos. 4,970,154 (13 Nov. 1990; Chang, BaylorCollege of Medicine), WO 96/40958 (19 Dec. 1996; Smith et al., BaylorCollege of Medicine) 5,679,559 (21 Oct. 1997; Kim et al., University ofUtah) 5,676,954 (14 Oct. 1997; Brigham, Vanderbilt University), and5,593,875 (14 Jan. 1997; Wurm et al., Genentech).

Another means to increase endogenous DKR polypeptide expression in acell via gene therapy is to insert one or more enhancer elements intothe DKR polypeptide promoter, where the enhancer element(s) can serve toincrease transcriptional activity of the DKR polypeptides gene. Theenhancer element(s) used will be selected based on the tissue in whichone desires to activate the gene(s); enhancer elements known to conferpromoter activation in that tissue will be selected. For example, if aDKR polypeptide is to be “turned on” in T-cells, the lck promoterenhancer element may be used. Here, the functional portion of thetranscriptional element to be added may be inserted into a fragment ofDNA containing the DKR polypeptide promoter (and optionally, vector, 5′and/or 3′ flanking sequence, etc.) using standard cloning techniques.This construct, known as a “homologous recombination construct” can thenbe introduced into the desired cells either ex vivo or in vivo.

Gene therapy can be used to decrease DKR polypeptide expression bymodifying the nucleotide sequence of the endogenous promoter(s). Suchmodification is typically accomplished via homologous recombinationmethods. For example, a DNA molecule containing all or a portion of thepromoter of the DKR gene(s) selected for inactivation can be engineeredto remove and/or replace pieces of the promoter that regulatetranscription. Here, the TATA box and/or the binding site of atranscriptional activator of the promoter may be deleted using standardmolecular biology techniques; such deletion can inhibit promoteractivity thereby repressing transcription of the corresponding DKR gene.Deletion of the TATA box or transcription activator binding site in thepromoter may be accomplished by generating a DNA construct comprisingall or the relevant portion of the DKR polypeptide promoter(s) (from thesame or a related species as the DKR gene(s) to be regulated) in whichone or more of the TATA box and/or transcriptional activator bindingsite nucleotides are mutated via substitution, deletion and/or insertionof one or more nucleotides such that the TATA box and/or activatorbinding site has decreased activity or is rendered completely inactive.This construct, which also will typically contain at least about 500bases of DNA that correspond to the native (endogenous) 5′ and 3′flanking regions of the promoter segment that has been modified, may beintroduced into the appropriate cells (either ex vivo or in vivo) eitherdirectly or via a viral vector as described above. Typically,integration of the construct into the genomic DNA of the cells will bevia homologous recombination, where the 5′ and 3′ flanking DNA sequencesin the promoter construct can serve to help integrate the modifiedpromoter region via hybridization to the endogenous chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit one or more DKR polypeptides. For example, antisense DNA or RNAmolecules, which have a sequence that is complementary to at least aportion of the selected DKR polypeptide gene(s) can be introduced intothe cell. Typically, each such antisense molecule will be complementaryto the start site (5′ end) of each selected DKR gene. When the antisensemolecule then hybridizes to the corresponding DKR polypeptides mRNA,translation of this mRNA is prevented.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more of the DKR polypeptides. Inthis situation, the DNA encoding a mutant full length or truncatedpolypeptide of each selected DKR polypeptide can be prepared andintroduced into the cells of a patient using either viral or non-viralmethods as described above. Each such mutant is typically designed tocompete with endogenous polypeptide in its biological role.

Samples of the E. coli cell lines GM121 and GM94 have been depositedwith the American Type Culture Collection, 10801 University Blvd.,Manassas, Va., USA on DATE as accession numbers X and Y, respectively.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLES Example 1 Cloning of the Mouse DKR-3 Gene

About 120 adult mice with an average body weight of about 18 grams wereeach injected intraperitoneally with a kainate solution (prepared as astock solution of about 1 mg/ml kainate in sterile PBS) at a dose ofabout 25 mg kainate per kilogram body weight. About six hours afterinjection, the mice were sacrificed, and the hippocampus was dissectedfrom each mouse. Total RNA was extracted from hippocampal tissue usingthe Trizol method (Gibco BRL, Grand Island, N.Y.). The poly(A+) mRNAfraction was isolated from total RNA using Message Maker (Gibco BRL,Grand Island, N.Y.) according to the manufacturer's recommendedprocedure. Hippocampal tissue was also obtained from control mice (whichreceived an injection of PBS only), and poly(a+) mRNA was obtained fromthis tissue as well using the same procedures.

Two random primed cDNA libraries were prepared; one from thekainate-treated and one from the control poly (A+) mRNA using theSuperscript® plasmid system (Gibco BRL, Gaithersburg, Md.). A randomcDNA primer containing an internal NotI restriction site was used toinitiate first strand synthesis and had the following sequence:

(SEQ ID NO:15) GGAAGGAAAAAAGCGGCCGCAACANNNNNNNNNwhere N is A, G, C, or T.

Both first strand cDNA synthesis and second strand cDNA synthesis wereperformed according to the manufacturer's recommended protocol. Aftersecond strand synthesis, the reaction products were extracted withphenol:chloroform:isoamyl alcohol (in a volume ratio of 25:24:1),followed by ethanol precipitation. The double strand cDNA products wereligated using standard ligation procedures to the following doublestranded oligonucleotide adapter (obtained from Gibco BRL, Grand Island,N.Y.):

(SEQ ID NO:16) TCGACCCACGCGTCCG (SEQ ID NO:17) GGGTGCGCAGGC

After ligation, the cDNA was digested to completion with NotI, and sizefractionated on a 1 percent agarose gel. The cDNA products between about250 and 800 base pairs were selected and purified from the gel using theQiagen® gel extraction kit (Qiagen, Chatsworth, Calif.). The purifiedcDNA products were directionally ligated into the vector pYY41L(American Type Culture Collection, “ATCC”; 10801 University Blvd.,Manassas, Va., USA; accession number 209636) which had been previouslydigested with NotI and SalI. The ligated cDNA was then introduced intoelectrocompetent ElectroMax® DH108 E. coli cells (Gibco-BRL, GrandIsland, N.Y.) via standard electroporation techniques. The library wasthen titered by a serial dilution of the transformation cell mixture.

About one million primary clones were divided into 20 pools (50,000clones each pool) and each pool was plated on 245 mm×245 mm square platecontaining MR2001 medium (MacConnel Research, San Diego, Calif.) andabout 60 ug/ml carbonocillin. After incubation overnight at 37 C, thecolonies were scraped off the plate in about 20 ml SOC (SOC containsabout 2 percent Bactotryptone, 0.5 percent yeast extract, 10 mM sodiumchloride, 2.5 mM potassium chloride, and 10 mM magnesium sulfate) andwere pelleted by centrifugation at about 6000 rpm for about 10 minutes.The plasmids were then recovered from the cells using Qiagen® maxi prepcolumns (Qiagen, Chatsworth, Calif.) according to the protocol suggestedby the manufacturer.

About two hundred and fifty thousand clones (50 ug total plasmids/10 ugfrom each pool) were used to transform yeast strain YPH499 (ATCCaccession number 90834) and an amylase-based signal trap assay wasconducted as follows (see co-pending U.S. Ser. No. 09/026,959 filed 20Feb. 1998 for a detailed description of this technique). Around 1000transformants were plated on a single starch-containing selection plate(15 cm diameter with a medium containing about 0.6 percent yeastnitrogen base, 2 percent glucose, 0.1 percent CAA, 1.0× trp dropoutsolution, 0.7 percent potato starch azure, and 1.5 percent agarose). Theplates were incubated at about 30 C for 4-5 days until full developmentof halos was observed. The colonies in the center of the halo werepicked and restreaked on a fresh plate to form single colonies. Thesingle colonies with halos were then picked and arrayed into 96 wellmicrotiter plates containing about 100 ul of water per well, therebygenerating the “yeast colony solutions”.

About ten microliters of each well of each yeast colony solution wasused as template to recover the cDNA fragment from that colony throughPCR. Therefore, ninety-six PCR reactions were independently performedusing PCR-Ready Beads® (96 well format, Amersham-Pharmacia Biotech,Pistcataway, N.J.) and the following oligonucleotides according to themanufacturer's protocol:

(SEQ ID NO:18) ACTAGCTCCAGTGATCTC (SEQ ID NO:19) CGTCATTGTTCTCGTTCC

PCR was conducted using a Perkin-Elmer 9600 thermocycler with thefollowing cycle conditions: 94 C for 10 minutes followed by 35 cycles of94 C for 30 seconds, 55 C for 30 seconds and 72 C for 1 minute, afterwhich a final extension cycle of 72 C for 10 minutes was conducted. MostPCR reactions contained a single PCR product. The amplified cDNAproducts were purified using the Qiagen® PCR purification kit (Qiagen,Chatsworth, Calif.). These products were sequenced on an AppliedBiosystems 373A automated DNA sequencer using the followingoligonucleotide primer:

(SEQ ID NO:35) GCTATACCAAGCATACAATC

Taq dye-terminator sequencing reactions (Applied Biosystems, FosterCity, Calif.) were conducted following the manufacturer's recommendedprocedures.

Each PCR fragment was translated in all six possible ways to identifythose fragments which (1) had a potential signal peptide in the samedirection as reporter gene; (2) had a stop codon(s) upstream of theputative methionine translation start site; and (3) appeared to lack atransmembrane domain.

One clone that met these criteria, termed “ymrs2-00009-c4”, was selectedfor further analysis. This clone contained 5′ sequence, including aputative signal sequence, but was lacking 3′ sequence.

To obtain the 3′ sequence of this clone, a 3′ RACE reaction wasperformed using as a template pool number 4 from the YmHK2 cDNA library.This YmHK2 library was prepared as follows: First strand cDNA synthesiswas performed using about 2 micrograms of the RNA obtained from thehippocampus of the kainate treated mice and about 1 ug of Not Iprimer-adapter having the following sequence:

(SEQ ID NO:42) GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT

Both the first strand and second strand cDNA synthesis reactions wereperformed using the Superscript® plasmid system (Gibco BRL, GrandIsland, N.Y.). After second strand synthesis, the double stranded cDNAproducts were ligated into the double stranded adapters of SEQ ID NOs:16and 17.

After ligation, the cDNA was digested to completion with Not I, and sizefractionated on a 0.8 percent agarose gel. The cDNA products larger thanabout 800 base pairs were selected and purified from the gel using theQiagen® gel extraction kit (Qiagen, Chatsworth, Calif.). The purifiedcDNA products were directly ligated into Sal I and Not I digestedpSport® vector (Gibco BRL, Grand Island, N.Y.).

The ligated cDNA products were then introduced into electrocompetent E.coli cells called ElectroMax® DH10B (Gibco BRL, Grand Island, N.Y.). Thelibrary was then titered.

About twelve million primary clones were obtained, and expanded intoabout 250 ml of LB containing about 100 ug/ml ampicillin. Afterovernight incubation at 37 C, the plasmids were recovered using theQiagen® maxi-prep kit (Qiagen, Chatsworth, Calif.).

About 20 ng of the plasmid library were used to transform theElectroMax® DH10B electrocompetent E. coli cells using standardelectroporation techniques. About two million transformants were dividedinto 40 pools (containing approximately 50,000 plasmids/pool). Each poolwas then expanded into about 3 ml of LB medium containing about 100ug/ml ampicillin. After overnight incubation at 37 C, the plasmids wererecovered using the Qiagen® mini-prep kit. The DNA from each pool werethen stored at about minus 20 C for future use.

The 3′ RACE reaction was performed using about 1.5 ng of pool #4 of theYmHK2 library as a template, and using the Advantage® cDNA PCR kit(Clontech, Palo Alto, Calif.) with the following oligonucleotides:

(SEQ ID NO:20) CCAGCTGCTCTGTGGCAGCCCAG (SEQ ID NO:21)CCCAGTCACGACGTTGTAAAACGACGGCC

The reaction was conducted in a standard thermocycler (Perkin-Elmer9600) for thirty five cycles under the following conditions: 94 C for 1minute; 94 C for 5 seconds, and 72 C for 5 minutes. This was followed bya final extension at 72 C for 10 minutes. About one microliter of thereaction products was diluted to 50 ul using TE buffer (10 mM TRIS pH8.0 and 1 mM EDTA).

To enrich the RACE reaction for the gene of interest, a nested PCRreaction was conducted using about five microliters of the TE solution(containing the RACE reaction products as described in the precedingparagraph) together with the following oligonucleotides:

(SEQ ID NO:22) AACATGCAGCGGCTCGGGGG (SEQ ID NO:23)GGTGACACTATAGAAGAGCTATGACGTCGC

The nested PCR reaction was incubated in a thermocycler (Perkin-Elmer9600) using the following protocol: 94 C for one minute; five cycles of94 C for 5 seconds followed by 72 C for 5 minutes; five cycles of 94 Cfor five seconds, followed by 70 C for 5 minutes; and 20-25 cycles of 94C for 5 seconds followed by 68 C for 5 minutes. After this PCR, the 3′RACE products and the nested PCR products were analyzed using standardagarose gel electrophoresis.

A PCR product of about 3.3 kb was identified from the nested PCRreaction. This fragment was purified using Qiagen® Gel Extraction Kit(Qiagen, Chatsworth, Calif.) and ligated into the vector pCRII-TOPO(Invitrogen, Carlsbad, Calif.) according to the procedures recommendedby the manufacturer. After ligation, the products were transformed intoOne Shot® E. coli cells (Invitrogen, Carlsbad, Calif.) and plated on aLB (Luria broth) plate containing about 100 ug/ml ampicillin and about1.6 mg X-gal. After overnight incubation at 37 C, 12 white colonies andone blue colony were selected, and screened using PCR-Ready Beads®(Amersham-Pharmacia Biotech, Pistcataway, N.J.) according to themanufacturer's recommended protocol using oligonucleotide SEQ ID NO:20together with the following primer:

(SEQ ID NO:24) GTGCTGAGTGTCTTCCATCAGC

Two colonies were picked that had yielded PCR products of the expectedsize of about 192 base pairs. These colonies were inoculated into about3 ml of LB medium containing about 100 ug/ml ampicillin, and wereincubated at 37 C. The cultures were placed on a shaker for about 16hours, and the plasmids were recovered using Qiagen® mini prep columns(Qiagen, Chatsworth, Calif.) according to the manufacturer's protocol.Plasmid DNA was then sequenced as described above.

A contiguous stretch of DNA of about 3366 nucleotides was assembled bycombining the sequence of clone ymrs2-00009-c4 (containing 5′ sequence)together with the nested PCR fragment of 3.3 kb containing 3′ sequence.Within this contiguous sequence is an open reading frame of 349 aminoacids. The nucleotide sequence of this novel mouse gene, referred to asDKR-3, is set forth in FIG. 1. The putative amino acid sequence, astranslated from the DNA sequence, is set forth in FIG. 8

A BLAST search of the Genbank database using the amino acid sequence ofDKR-3 revealed that this open reading frame has homology to a gene knownas human rig-like 7-1 mRNA (Genbank accession number AF034208; see alsoLigon et al., J NeuroVirology, 4:217-226 [1998]). DKR-3 also hashomology to the gene for chicken lens fiber protein clfest4 (Genbankaccession number D26311); the overall identity to this protein is about50 percent with the highest homology in the middle of the protein.

Example 2 Cloning of the Human DKR-3 Gene

Mouse DKR-3 DNA can be used to search a public EST database for humanhomologs, resulting in the identification of the following Genbankaccession numbers:

AA628979

AA349552

AA633061

AA351624

W61032

T30923

AA683017

AA324686

T08793

T31076

R14945

AA226979

W45085

AA424460

R58671

R57834

AF034208

These EST sequences were analyzed and assembled to create a putativesequence for human DKR-3. Based on this putative sequence, twooligonucleotides were designed for use in PCR in an attempt to clone thehuman DKR-3 gene. The sequence of these oligonucleotides is:

(SEQ ID NO:25) GAGATGCAGCGGCTTGGGGCCACCC (SEQ ID NO:26)GCCTGGTCAGCCCACGCCTAAAG

PCR was performed using the Advantage® cDNA PCR kit (Clontech, PaloAlto, Calif.) together with human fetal brain Quick-Clone® cDNA(Clontech). PCR was conducted in a thermocycler (Perkin-Elmer 9600)under the following cycle conditions: 94 C for 2 minute; 94 C for 30seconds, and 72 C for 2 minutes. Thirty-five cycles were conducted afterwhich samples were treated at 72 C for 10 minutes. A single fragment ofabout 1150 base pairs was visible when the PCR products were visualizedon a 1 percent agarose gel. This fragment was purified using the Qiagen®Gel Extraction Kit (Qiagen, Chatsworth, Calif.) and ligated into thevector pCRII-TOPO (Invitrogen, Carlsbad, Calif.). After ligation, theproducts were transformed into One Shoot E. coli® (Invitrogen, Carlsbad,Calif.) and plated on a LB plate containing about 100 ug/ml ampicillinand about 1.6 mg X-gal. After overnight incubation at 37 C, 2 whitecolonies were picked and inoculated into about 3 ml of LB mediumcontaining about 100 ug/ml ampicillin. The cultures were kept on ashaker at about 37 C for about 16 hours. The plasmids were isolatedusing Qiagen® mini-prep columns (Qiagen, Chatsworth, Calif.) accordingto the manufacturer's recommended protocol, and the inserts were thensequenced using methods described above.

The cloned fragment is 1141 bp in length and contains an open readingframe of 350 amino acids. The nucleotide sequence is set forth in FIG.2, and the putative amino acid sequence, as translated from the DNAsequence, is set forth in FIG. 9. This amino acid sequence is about 80percent identical to the mouse DKR-3 gene. In addition, human DKR-3 isidentical to the human rig-like protein fragment described by Lignon etal., supra between amino acids 157 and 308 of DKR-3. Significantly, therig-like protein has an amino terminal start corresponding to amino acid156 of DKR-3. Rig-like does not appear to be a secreted protein, and thecarboxy terminal region of rig-like protein has no homology to humanDKR-3. Just as for mouse DKR-3, human DKR-3 is about 54 percentidentical to the chicken lens fiber protein clfest. Human DKR-3 appearsto be secreted, with a signal peptide cleavage site after either aminoacid 20 or 21. Other potential cleavage sites (due to signal peptides orto other endogenous processing sites are after amino acid 16, 22, 32,and/or 41). There appear to be N-linked glycosylation sites at aminoacids 96, 106, 121, and 204, which would render them preferable sitesfor generating substitution mutants. Human DKR-3 and mouse DKR-3 aminoacid sequences differ at amino acid positions 6, 7, 11, 24, 27, 29, 30,32, 33, 39, 81, 89, 93, 99, 101, 103, 109, 113, 115, 123, 126, 142, 156,157, 162, 165, 173, 175, 191, 197, 198, 201, 203, 245, 247, 259, 283,287, 292, 294, 295, 296, 298, 299, 304, 310, 311, 312, 314, 315, 329,330, 334, 335, 336, 339, 340, 341, 342, 343, 345, and 347 (all withrespect to the human DKR-3 sequence), which renders these positionspreferable for generating human DKR-3 substitution or deletion variants.Based on computer analysis of the amino acid sequence of DKR-3,significant regions of the molecule include the span from about aminoacids 21-145 (a potential alpha-helical region and region of potentialN-linked glycosylation) such as for example amino acids 21-145, 40-145,40-150, 45-145, and 45-150, the span from about amino acids 145-350,such as, for example 145-290, 145-300, and 145-350, and the span fromabout amino acids 300-350 (a second potential alpha-helical region),such as for example amino acids 310-350. Such regions would be suitablefragments of full length DKR-3.

Northern blot analysis was conducted to assess the tissue specificexpression of human DKR-3. A probe for use in Northern blot analysis wasprepared by PCR of human fetal brain Quick-Clone® cDNA (Clontech, PaloAlto, Calif.) using the following oligonucleotides:

(SEQ ID NO:27) CCTGCTGCTGGCGGCGGCGGTCCCCACGGC (SEQ ID NO:28)GCCTGGTCAGCCCACGCCTAAAG

The PCR reaction was conducted in a thermocycler (Perkin-Elmer 9600).PCR conditions were: 94 C for 2 minute; 94 C for 30 seconds, and 72 Cfor 2 and ½ minutes. Thirty-five cycles were conducted followed by afinal extension treatment at 72 C for 10 minutes. PCR products were runon a one percent agarose gel, and a band of about 1100 bp was gelpurified using the Qiagen gel extraction kit (Qiagen®, Chatsworth,Calif.), cloned into the vector CRII-TOPO (Invitrogen, Carlsbad, Calif.)and sequenced to confirm that the band contained the human DKR-3 openreading frame minus the amino terminal 10 amino acids.

About twenty-five nanograms of this probe was denatured by heating toabout 100 C for about 5 minutes, followed by placing on ice, and thenradioactively labeled with alpha-32P-dCTP using the Rediprime® labelingkit (Amersham, Arlington Heights, Ill.) and following the manufacturer'sinstructions. A human multiple tissue Northern blot was purchased(Clontech, Palo Alto, Calif.) and was first prehybridized in about 5 mlof Clontech Express® hybridization buffer at about 68 C for 30-60minutes. After prehybridization, the labeled probe was added to thesolution and allowed to hybridize for about 60 minutes. Afterhybridization, the blot was first washed with 2×SSC plus 0.05 percentSDS at room temperature for about 30 minutes, then washed with 0.1×SSCplus 0.1 percent SDS at about 65 C for about 30 minutes. The blot wasdried briefly and then exposed to a Phosphorimager screen (MolecularDynamics, Sunnyvale, Calif.). After overnight exposure, the image of theblot was analyzed on a Storm 820 machine (Molecular Dynamics, Sunnyvale,Calif.) with Imagequat software (Molecular Dynamics, Sunnyvale, Calif.).

The size of the human DKR-3 RNA transcript is about 2.6 kb. The resultsof the Northern blot analysis indicate that human DKR-3 is highlyexpressed in adult heart and brain, although weak expression inplacenta, adult lung, skeletal muscle, kidney, and pancreas is alsoapparent. A second smaller transcript is apparent in adult pancreas, andcould result from degradation of the full length transcript.

To evaluate the role of this gene in cancer, a variety of human cancercell lines were analyzed for the presence or absence of DKR-3 RNAtranscript.

The glioblastoma cell lines Hs 683; A 172; SNB-19; U-87MG; and U-373MGare all from ATCC, and cultured in the media recommended by ATCC.

Normal human mammary epithelial cells (NMECs) derived from reductionmammoplasties were purchased from Clonetics Corp. (San Diego, Calif.)and the Corriel Institute (Camden, N.J.). The immortalized breastepithelial cell line MCF-10 and the ER+ cell line MCF-7 can be obtainedfrom the American Type Culture Collection. The ER+ BT20T cells wereprovided by Dr. K. Keyomarsi (N.Y. State Dept. of Health). Immortalized184A1 and other breast cancer cells including T47-D, ZR75-1, and BT474,MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-453, MD-MBA-468, HS578T andSKBr3 were all obtained from the American Type Culture Collection (10801University Blvd., Manassas, Va.).

NMECs, 184A1 and MCF10 cells were cultured in a modified DME/F12 medium(Gibco/BRL, Grand Island, N.Y.) supplemented with 10 mM Hepes, 2 mMglutamine, 0.1 mM nonessential amino acids, 0.5 mM ethanolamine, 5 mg/mltransferrin, 1 mg/ml Bovine serum albumin, 5.0 ng/ml sodium selenite, 20ng/ml triiodothyronine, 10 ng/ml EGF, 5 μg/ml insulin and 0.5 μg/mlhydrocortisone (DMEM/F12C) (Ethier et al, Cancer Letters, 74:189-195[1993]). The ER+ and ER+ breast cancer cells were cultured in Alpha orRichter improved minimal essential medium (MEM) (Gibco/BRL) supplementedwith 10 mM Hepes, 2 mM glutamine, 0.1 mM nonessential amino acids, 10percent fetal bovine serum and 1 μg/ml insulin.

Normal human bronchial and cervical epithelial cells were purchased fromClonetics Corp. (San Diego, Calif.). Normal cervical epithelial cellswere culture in KBM2 (Clonetics Corp. San Diego, Calif.) supplementedwith 13 mg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 2ng/ml EGF, 0.5 mg/ml epinephrine, 0.1 ng/ml retinoic acid, 5 μg/mltransferrin, 6.5 ng/ml triiodothyronine and 5 μg/ml insulin. Normalbronchial epithelial cells were cultured in BEBM (Clonetics Crop., SanDiego, Calif.) supplemented with 0.5 mg/ml hydrocortisone, 0.5 ng/mlEGF, 0.5 μg/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 0.1ng/ml retinoic acid and 5.5 ng/ml triiodthyronine.

The lung cancer cell lines H1299, H23, H358, H441, H460, H520, H522,H727, H146, H209, H446, H510A, H526, and H889 and the cervical cancercells Caski, C-4-I, MS751, SiHa and C-33-A were all obtained from theAmerican Type Culture Collection. The lung cancer cells were cultured inRPMI (MEM) (Gibco/BRL) supplemented with 10 mM Hepes, 2 mM glutamine,0.1 mM nonessential amino acids and 10 percent fetal bovine serum (FBS).The cervical cancer cells were cultured in Earles MEM supplemented with0.1 mM nonessential amino acids, 1 mM sodium pyruvate and 10 percentFBS. All cells were routinely screened for mycoplasma contamination andmaintained at about 37° C. in an atmosphere of about 6.5 percent CO₂.

Total RNA was prepared by lysing cell monolayers in guanidiniumisothiocyanate and centrifuging over a 5.7 M CsCl cushion as describedpreviously (Gudas, Proc. Natl. Acad. Sci. USA, 85:4705-4709 [1988]). RNA(about 20 ug) was electrophoresed on denaturing formaldehyde gels,transferred to MagnaNT membranes (Micron Separations Inc., Westboro, Ma)and cross-linked with UV irradiation.

The blots were prehybridized, probed, and washed under the sameconditions as those set forth above for the tissue blot. The blots weredried briefly and then exposed to a Phosphorimager screen (MolecularDynamics, Sunnyvale, Calif.). After overnight exposure, the image of theblot was analyzed on a Storm 820 machine with Imagequat software (bothfrom Molecular Dynamics).

The results are shown in FIGS. 15A-15D. As can be seen in FIG. 15A,expression of DKR-3 is decreased in most of the breast cancer cell linesas compared to the normal cell lines. FIG. 15B indicates that DKR-3expression is decreased in the non-small cell lung cancer cell lines,and in most of the small cell lung cancer cell lines as well. FIG. 15Cindicates that expression of DKR-3 is decreased in three glioblastomacell lines (SNB-19, U-87MG, and U-373MG) that are capable of formingtumors in nude mice (the other two cell lines, Hs 683 and A 172 do notform tumors in nude mice). FIG. 15D indicates that expression of DKR-3is reduced in cervical cancer cell lines as compared to normal andimmortalized cells.

Example 3 Cloning of the Human DKR-1 Gene

Human and mouse DKR-3 cDNA and amino acid sequences were used to searchGenbank using the BLAST program in an attempt to identify DKR-3 relatedgenes. A number of EST (expressed sequence tags) were found and wereanalyzed to determine whether the sequences overlapped. Using thefollowing human EST accessions, a novel gene, termed DKR-1, waspredicted.

AA336797

R27865

W39690

AA043027

HUM517H04B

AA143670

W51876

N94525

AA641247

AA137219

AA115249

AA031969

AA136192

AA032060

AA035583

AA207078

AA371363

AA037322

AA088618

W46873

AA115337

AA693679

W30750

H83554

PCR was conducted in an attempt to clone the full length gene, and thefollowing two oligonucleotides were used for PCR:

CCCGGACCCTGACTCTGCAGCCG (SEQ ID NO:29) GAGGAAAAATAGGCAGTGCAGCACC (SEQ IDNO:30)

PCR was performed using the Advantage® cDNA PCR kit (Clontech, PaloAlto, Calif.) containing the oligonucleotides listed above and humanplacenta Quick-Clone® cDNA (Clontech, Palo Alto, Calif.). The reactionwas conducted according to the manufacturer's recommendations.Thirty-five cycles of PCR were conducted in a thermocycler (Perkin-Elmer9600) under the following conditions: 94 C for 2 minutes; 94 C for 30seconds, and 72 C for 1½ minutes, followed by a final extension at 72 Cfor 10 minutes.

After cycling, the PCR products were analyzed on a one percent agarosegel. A single band of about 1200 base pairs in length was detected afteragarose gel electrophoresis. This fragment was purified using theQiagen® gel extraction kit (Qiagen, Chatsworth, Calif.) and ligated intothe vector pCRII-TOPO (Invitrogen, Carlsbad, Calif.) using standardligation procedures. After ligation, the products were transformed intoOne Shoot® competent E. coli cells according to the proceduresrecommended by manufacturer (Invitrogen, Carlsbad, Calif.). Thetransformed E. coli cells were plated on a LB plate containing about 100ug/ml ampicillin and about 1.6 mg X-gal.

After overnight incubation at about 37 C, two white colonies were pickedand inoculated into about 3 ml of TB containing 100 ug/ml ampicillin.The culture was incubated at about 37 C for about 16 hours, plasmidswere then recovered using Qiagen® mini-prep columns (Qiagen, Chatsworth,Calif.) and sequenced. Both colonies contained the same insert.

The insert is 1193 base pairs, and is referred to as human DKR-1. Thesequence of this gene is set forth in FIG. 3. This gene contains an openreading frame of 266 amino acids. The amino acid sequence is set forthin FIG. 10. A stop codon is present upstream of the first methionine,indicating the first methionine is likely to be the amino terminus ofthe protein. Human DKR-1 has a predicted signal peptide with a predictedsignal peptide cleavage site between amino acids 19 and 20.

The gene has about 80 percent homology to the mouse gene dkk-1 (Glinkaet al., supra), however the mouse dkk-1 gene is 272 amino acids inlength while human DKR-1 is 266 amino acids in length. Human DKR-1differs from mouse dkk-1 at amino acid positions 3, 4, 5, 7, 8, 10, 12,13, 14, 15, 16, 17, 18, 19, 22, 23, 24, 29, 53, 55, 62, 66, 69, 77, 93,98, 101, 105, 106, 123, 139, 140, 143, 144, 153, 155, 157, 158, 163,164, 165, 169, 175, 178, 197, 224, and 244. In addition, the alignmentof human DKR-1 and mouse dkk-1 shows one gap in human DKR-1 betweenamino acids 37 and 38, and two gaps between 103 and 104, 146 and 147,and 165 and 166. Glinka et al. state on page 362 of their article that“Coordinates of Xenopus dkk family members have been deposited inGenbank with the following accession numbers . . . hdkk-1 AA207078.”However, forward three frame translations of AA207078 by the inventorsherein showed no homology to the published mouse and Xenopus dkk-1sequences, or to the human DKR-1 sequence, except in the 3′ end of thisaccession, which exhibits a 95 percent identity to human DKR-1 fromamino acids 81-179, indicating that AA207078 does not encode full lengthhuman dkk-1. Significantly, AA207078 is missing amino acids 1-90 and180-350 of human DKR-1 which includes the signal peptide and the secondcysteine right domain respectively.

Example 4 Cloning of the Mouse DKR-2 Gene

Genbank accession number AA265561 (a mouse sequence) has homology toboth human DKR-1 and human DKR-3 at the amino acid level based primarilyon its cysteine pattern.

To extend this EST sequence in both the 5′ and 3′ directions, thefollowing oligonucleotides were designed:

GCCACAGTCCCCACCAAGGATCATC (SEQ ID NO:31) GATGATCCTTGGTGGGGACTGTGGC (SEQID NO:32) CTGCAAACCAGTGCTCCATCAGGG (SEQ ID NO:33)CCCTGATGGAGCACTGGTTTGCAG (SEQ ID NO:34)

Separately, 5′ RACE and 3′ RACE reactions were performed according tothe manufacturer's protocol using mouse heart Marathon-Ready® cDNA andthe Advantage® cDNA PCR kit (both from Clontech, Palo Alto, Calif.) andusing oligonucleotide SEQ ID NOs: 31 and 34. The RACE reactions wereincubated in a thermocycler (Perkin-Elmer 9600) using the followingcycling conditions: 94 C for one minute; five cycles of 94 C for 5seconds followed by 72 C for 5 minutes; five cycles of 94 C for fiveseconds, followed by 70 C for 5 minutes; and 20-25 cycles of 94 C for 5seconds followed by 68 C for 5 minutes.

To enrich each RACE reaction for the desired product, about onemicroliter of each of the RACE PCR products was added together, and themixture was diluted to about 50 ul using TE buffer. About fivemicroliters of this solution were used to conduct nested PCR reactions.The Advantage® cDNA PCR kit (Clontech, Palo Alto, Calif.) andoligonucleotide SEQ ID NOs: 32 and 33 were used for the 5′ and 3′nesting reactions, respectively. The nested PCR reactions were incubatedin a thermocycler (Perkin-Elmer 9600) using the following program forthirty five cycles: 94 C for 1 minute; 94 C for 5 seconds; and 72 C for2 minutes. A final extension was then conducted at 72 C for 10 minutes.The PCR products were analyzed using a one percent agarose gel.

Several fragments ranging from about 500 bp to about 1500 base pairswere obtained from the 5′ nested PCR reaction, and two fragments ofabout 1900 bp and 450 bp were obtained from the 3′ nested PCR reaction.These PCR products were purified using the Qiagen® PCR purification kit(Qiagen, Chatsworth, Calif.) and were then ligated into the vectorpCRII-TOPO (Invitrogen). The ligation products were transformed intoOneShot® E. coli cells (Invitrogen, Carlsbad, Calif.), and the cellswere then plated on to two X-gal containing plates (one for eachreaction) as described above.

Eight white colonies from each plate were picked and PCR selected viaRACE reactions using the Clontech primer AP2 and the oligonucleotide SEQID NO:32 (for the 5′ RACE) or the oligonucleotide SEQ ID NO:33 (for the3′ RACE). Three colonies from each plate that contained the correct sizefragments were cultured, and the plasmids were isolated and sequencedusing procedures described above.

Three clones, 9813302, 9813304 and 9813305 contained sequence whichextended the EST sequence in the 5′ direction. One clone, 9813308,contained sequence which extended the EST sequence in the 3′ direction.A continuous sequence of 2678 base pairs was thus assembled using thesequence of clones 9813308, 9813304, and the EST AA265561. This fulllength DNA has been termed DKR-2, and the sequence is set forth in FIG.4. The corresponding amino acid sequence is set forth in FIG. 11.

Within the amino acid sequence is an open reading frame of 259 aminoacids. This protein has approximately 38 percent identity with mousedkk-1 at the amino acid level. Mouse DKR-2 has a predicted signalpeptide with a signal peptide cleavage site between amino acids 33 and34.

Example 5 Cloning of the Human DKR-2 Gene

The Genbank EST database was searched using the BLAST program with bothDNA and amino acid sequences from human DKR-1 and human DKR-3, and onehuman EST, W55979, was identified that showed homology to both humanDKR-1 and human DKR-3 at the amino acid level based on its cysteinepattern. W55979 is about 88 percent identical to mouse DKR-2 at the DNAlevel, and about 93 percent identical to mouse DKR-2 at the amino acidlevel.

A BLAST search of Genbank W55979 indicated that W55979 has homology toBAC clone number B284B3 (Genbank accession number AC003099). BAC cloneB284B3 is 95129 base pairs in length. Three portions of W55979 arehomologous to three different regions of BAC clone B284B3, indicatingthat human DKR-2 has at least three exons. A 3′ sequence of 556 bp inlength was assembled based on the sequences of both BAC clone B284B3 andW55979, and it was determined that this sequence is the 3′ portion ofthe human ortholog of mouse DKR-2. Within this 3′ sequence of humanDKR-2 is an open reading frame of 174 amino acids, and a stop codon ispresent after amino acid 174. This 3′ sequence of human DKR-2 is about97 percent identical to mouse DKR-2.

To obtain the 5′ end sequence of human DKR-2, a 5′ RACE reaction wasperformed using Clontech human heart Marathon-Ready® cDNA and theAdvantage® cDNA PCR kit, together with oligonucleotide SEQ ID NO:34. TheRACE reaction was performed according to the manufacturer's protocol.The 5′ RACE reaction products were then subjected to nesting PCR toenrich for the 5′ sequence using the Advantage® cDNA PCR kit andoligonucleotide SEQ ID NO:32. The PCR conditions for both the 5′ RACEreaction and the nested PCR reaction were the same as those described inExample 4.

The nested PCR products were purified using the Qiagen® (Qiagen,Chatsworth, Calif.) PCR purification kit, and were ligated into thevector Zero-Blunt® (Invitrogen, San Diego, Calif.) according to theprocedures recommended by the manufacturer. The ligation products weretransformed into OneShot® E. coli cells which were then plated on X-galcontaining plates as described above.

After overnight culturing, three white colonies were picked and wereinoculated into about 3 ml of TB containing about 100 ug/ml ampicillin.The cultures were allowed to grow for about 16 hours, after which theplasmids were isolated using Qiagen® mini-prep columns (Qiagen,Chatsworth, Calif.) according to the manufacturer's protocol. Thesequence of each insert was then obtained.

One of the 5′-RACE clones, termed 9812826, extended the human DKR-2sequence 5′-terminally. A contiguous sequence of 1531 bp in length wasassembled using this clone 9812826 together with the human DKR-2 3′sequence. Within this contiguous sequence is an open reading frame of259 amino acids. The human DKR-2 gene has a predicted signal peptide ofabout 33 amino acids, with a predicted cut site between amino acids 33and 34, and is about 95 percent identical to mouse DKR-2 at the aminoacid level. The amino acid positions that differ between human and mouseDKR-2 include (with respect to the numbering of the human sequence) 7,12, 28, 48, 50, 58, 71, 102, 119, 170, 173, and 191, rendering thesepositions preferable for generating amino acid substitution or deletionvariants.

An alternative spliced isoform of human DKR-2 was discovered when PCRwas conducted using human heart Marathon-Ready® cDNA (Clontech, PaloAlto, Calif.) and the Advantage® cDNA PCR kit (Clontech, Palo Alto,Calif.) together with the following oligonucleotides:

GGGTTGAGGGAACACAATCTGCAAG (SEQ ID NO:36) GTCTGCAATTGATGATGTTCCTCAATGG(SEQ ID NO:37)

PCR was conducted using parameters set forth in the manufacturer'sprotocol. PCR products were analyzed by agarose gel electrophoresis, andtwo PCR products were obtained. The bands corresponding to theseproducts were gel purified as described above, amplified and purified asdescribed above, and then sequenced. One product corresponded to fulllength DKR-2, however, the other band corresponded to an isoform ofDKR-2. This isoform has an open reading frame of 207 amino acids, andappears to be missing an exon. This isoform is referred to as humanDKR-2a. The DNA sequence of human DKR-2a is set forth in FIG. 6, and theamino acid sequence as translated from the DNA is set forth in FIG. 13.

Example 6 Cloning of the Human DKR-4 Gene

A human EST that showed significant homology to human DKR-1 and humanDKR-3 on protein level was identified in Genbank. This sequence, Genbankaccession number AA565546, has a cysteine pattern that is similar tothat of human DKR-1 and human DKR-3.

A BLAST search of Genbank showed no human ESTs overlapping withAA565546. Therefore, to extend the EST sequence in the 5′ direction, a5′ RACE reaction was performed using human heart Marathon-Ready®cDNA(Clontech, Palo Alto, Calif.) together with the Advantage® cDNA PCR kit(Clontech, Palo Alto, Calif.) and the following oligonucleotide:

CCAGGGCCACAGTCGCAACGCTGG (SEQ ID NO:38)

The RACE reaction was performed according to the protocol provided withthe Advantage® kit. After 5′ RACE, the products were nested to enrichfor the desired 5′ sequence using the Advantage® cDNA PCR kit accordingto the manufacturer's recommendations, together with the followingoligonucleotide:

CTCCCTCTTGTCCCTTCCTGCCTTG (SEQ ID NO:39)

After the nested PCR reaction, the products were purified using theQiagen® PCR purification kit (Qiagen, Chatsworth, Calif.), ligated intothe vector pCRII-TOPO (Invitrogen, Carlsbad, Calif.), and transformedinto OneShot® E. coli cells as described above. After transformation,the cells were plated on a LB plate containing about 100 ug/mlampicillin and about 1.6 mg X-gal.

After overnight incubation at 37 C, four white colonies were picked fromthe plate and were inoculated in about 3 ml TB containing about 100ug/ml ampicillin. The cultures were incubated at about 37 C for about 16hours. The plasmids were then recovered using Qiagen® mini-prep columns(Qiagen, Chatsworth, Calif.) and sequenced.

Two clones, termed 9813563 and 9853564, were found to contain the 5′sequence of human DKR-4.

To obtain the 3′ sequence of human DKR-4, a 3′ RACE reaction wasperformed using human uterus Marathon-Ready® cDNA (Clontech, Palo Alto,Calif.) together with the Advantage® cDNA PCR kit (Clontech) and thefollowing oligonucleotide:

CAAGGCAGGAAGGGACAAGAGGGAG (SEQ ID NO:40)

The 3′ RACE reaction was performed according to the manufacturer'srecommendations. After the RACE reaction, the products were nested usingthe Advantage® cDNA PCR kit and the following oligonucleotide:

CCAGCGTTGCGACTGTGGCCCTGG (SEQ ID NO:41)

The parameters for PCR were 94 C for 1 minute followed by thirty fivecycles of 94 C for 5 seconds and then 72 C for 2 minutes, after which afinal extension of 70 C for 10 minutes was conducted. After the nestingreaction, the products were analyzed on a 1 percent agarose gel. Asingle band of about 1200 bp in length was observed. This band waspurified from the gel using methods described above, and was then clonedinto the vector pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) andsequenced. Sequence of this band indicated that it contained the 3′sequence of human DKR-4, and this sequence was assembled together withthe 5′ sequence (from clones 9813563 and 9853564) to generated the fulllength sequence of human DKR-4. This sequence is set forth in FIGS. 7(DNA sequence) and 14 (translated amino acid sequence). The polypeptideis 224 amino acids in length and is about 34 percent identical to humanDKR-1 at the amino acid sequence level.

Example 7 Expression of Human DKR-1 in Bacteria

PCR amplification employing the primer pairs and template describedbelow were used to generate a recombinant form of human DKR-1. Oneprimer of each pair introduces a TAA stop codon and a unique BamHI sitefollowing the carboxy terminus of the gene. The other primer of eachpair introduces a unique NdeI site, a N-terminal methionine, andoptimized codons for the amino terminal portion of the gene. PCR andthermocycling was performed using standard recombinant DNA methodology.The PCR products were purified, restriction digested, and inserted intothe unique NdeI and BamHI sites of vector pAMG21 (ATCC accession no.98113) and transformed into the prototrophic E. coli host GM121(deposited with the American Type Culture Collection on XX as accessionnumber XX). Other commonly used E. coli expression vectors and hostcells are also suitable for expression by one skilled in the art. Aftertransformation, positive clones were selected and examined forexpression of the recombinant gene product.

The construct pAMG21-human DKR-1-24-266 was engineered to be 244 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-His-Pro-Leu-Leu-Gly (SEQ ID NO:43) Thr-Cys-Gln-Arg-His (SEQ IDNO:44)The template used for PCR was human DKR-1 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct:

(SEQ ID NO:45) GTTCTCCTCATATGCATCCATTATTAGGCGTAAGTGCCACCTTGAACTCGGTTCTCAAT (SEQ ID NO:46) TACGCACTGGATCCTTAGTGTCTCTGACAAGTGTGAAG

Transformed E. coli strain GM121 containing pAMG21-human DKR-1-24-266were grown in 2× YT media containing 20 micrograms/ml kanamycin at 30 Cuntil the culture reached an optical density of about 600 nm of about0.5. Induction of DKR-1 protein expression was achieved by addition ofVibrio fischeri synthetic autoinducer to 100 ng/ml final and incubationof the culture at either 30° C. or 37° C. for about 9 hours further withshaking. In addition, as a uninduced control, for each culture noautoinducer was added to an aliquot of the culture, but the culture wasalso incubated for about 9 hours further at about 30 C with shakingalong with the induced cultures. After about 9 hours, the opticaldensity of cultures were measured at 600 nm, an aliquot of cultures wereexamined by oil emersion microscopy at 1600× magnification, and aliquotsof cultures were pelleted by centrifugation. Bacterial pellets ofcultures were processed for SDS-polyacrylamide gel electrophoresis on a14 percent gelto examine levels of protein produced in crude lysates andfor N-terminal sequencing confirmation of the recombinant gene product.The gel was stained with Coomassie blue.

The results are shown in the photo of FIG. 16. Lane 1 contains molecularweight markers; Lanes 2 and 5 contain crude lysates of uninduced controlcells incubated at 30 C; Lanes 3 and 6 are crude lysates of inducedcells cultured at 30 C; Lanes 4 and 7 are crude lysates of induced cellscultured at 37 C. The arrow on the left of Lane 1 indicates the expectedlocation of human DKR-1-24-266. As can be seen, large amounts ofrecombinant protein were observed in crude lysates of induced culturesat both 30° C. and 37° C. (Lanes 3 and 6, and 4 and 7). Microscopicanalysis of bacterial cells revealed most cells contained at least oneinclusion body, suggesting that at least some of the protein may beproduced in the insoluble fraction of E. coli.

Example 8 Expression of DKR-2 in Bacteria

PCR amplification employing the primer pairs and templates describedbelow were used to generate various forms of DKR-2. One primer of eachpair introduces a TAA stop codon and a unique BamHI site following thecarboxy terminus of the gene. The other primer of each pair introduces aunique NdeI site, a N-terminal methionine, and optimized codons for theamino terminal portion of the gene. PCR and thermocycling was performedusing standard recombinant DNA methodology. The PCR products werepurified, restriction digested, and inserted into the unique NdeI andBamHI sites of vector pAMG21 (ATCC accession no. 98113) and transformedinto either prototrophic E. coli host GM121 or GM94 (GM 94 was depositedwith the ATCC on XX as accession number XX). Other commonly used E. coliexpression vectors and host cells are also suitable for expression.After transformation, positive clones were selected and examined forexpression of the recombinant gene product.

The construct pAMG21-human DKR-2-26-259 was engineered to be 235 aminoacids in length and have the following N-terminal and the followingC-terminal amino acids, respectively:

Met-Ser-Gln-Ile-Gly-Ser (SEQ ID NO:47) Val-Cys-Gln-Lys-Ile. (SEQ IDNO:48)

The template used for PCR was human DKR-2 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct.

(SEQ ID NO:49) GTTCTCCTCATATGTCTCAAATTGGTAGTTCTCGTGCCAAACTCAACTCC ATCAAG(SEQ ID NO:50) TACGCACTGGATCCTTAAATTTTCTGACACACATGGAGT

The construct pAMG21 mouse DKR-2-26-259 was engineered to be 235 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-Ser-Gln-Leu-Gly-Ser (SEQ ID NO:51) Val-Cys-Gln-Lys-Ile (SEQ IDNO:52)The template used for PCR was mouse DKR-2 cDNA, and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct.

(SEQ ID NO:53) GTTCTCCTCATATGTCTCAATTAGGTAGCTCTCGTGCTAAACTCAACTCCATCAAGTCC (SEQ ID NO:54) TACGCACTGGATCCTTAGATCTTCTGGCATACATGGAGT

Transformed E. coli GM121 or GM94 containing either pAMG21-humanDKR-2-26-259 or pAMG21-mouse DKR-2-26-259 plasmid were grown in 2× YTmedia containing 20 μg/ml kanamycin at 30° C. until the culture reachedan optical density at 600 nm of about 0.5. Induction of DKR-2 proteinexpression was achieved by addition of Vibrio fischeri syntheticautoinducer to 100 ng/ml final and incubation of the culture at either30 C or 37 C for about 5 or 9 hours further with shaking. In addition,as a uninduced control, for each culture no autoinducer was added to analiquot of the culture, but the culture was also incubated for about 5or 9 hours further at 30 C with shaking along with the induced cultures.After either 5 or 9 hours incubation, the optical density of cultureswere measured at about 600 nm, an aliquot of cultures were examined byoil emersion microscopy at 1600× magnification, and aliquots of cultureswere pelleted by centrifugation. Bacterial pellets of cultures wereprocessed for SDS-polyacrylamide gel electrophoresis on a 14 percent gelto examine levels of protein produced in crude lysates and forN-terminal sequencing confirmation of the recombinant gene product. Thegel was stained with Coomassie blue.

The results are shown in FIG. 16, Lanes 8-10 (human DKR-2 polypeptide)and in FIG. 17 (mouse DKR-2 polypeptide). In FIG. 16, Lane 8 containscrude lysate of uninduced control cells; Lane 9 contains crude lysate ofinduced cells cultured at 30 C, and Lane 10 contains crude lysate ofinduced cells cultured at 37 C. The arrow to the left of Lane 10indicates the expected location of human DKR-2-26-259. As can be seen,significant amounts of polypeptide were generated in the inducedcultures whether grown at 30 C or 37 C, while the uninduced cells didnot produce a large amount of polypeptide. FIG. 17 shows the results ofpolypeptide production of mouse DKR-2-26-259. Lane 1 is molecular weightmarkers. Lanes 2-4 are one clone of E. coli cells transfected with theDKR-2 plasmid, while Lanes 5-7 are a second clone transfected with thesame plasmid. Lanes 2 and 5 are crude lysates of uninduced controlcells; Lanes 3 and 6 are crude lysates of induced cells cultured at 30C; and Lanes 4 and 7 are crude lysates of cells cultured at 37 C. Thearrows to the left of Lanes 4 and 7 indicate the expected location ofthe DKR-2 polypeptide. As can be seen, large amounts of recombinantprotein were observed in crude lysates of induced cultures at 37 C butnot at 30 C. Microscopic analysis of bacterial cells revealed most cellscontained at least one inclusion body, suggesting that at least some ofthe protein may be produced in the insoluble fraction of E. coli.

Example 9 Expression of DKR-3 in Bacteria

PCR amplification employing the primer pairs and templates describedbelow were used to generate various forms of DKR-3. One primer of eachpair introduces a TAA stop codon and a unique SacII site following thecarboxy terminus of the gene. The other primer of each pair introduces aunique NdeI site, a N-terminal methionine, and optimized codons for theamino terminal portion of the gene. PCR and thermocycling was performedusing standard recombinant DNA methodology. The PCR products werepurified, restriction digested, and inserted into the unique NdeI andSacII sites of vector pAMG21 (ATCC accession no. 98113) and transformedinto the prototrophic E. coli host GM121. Other commonly used E. coliexpression vectors and host cells are also suitable for expression byone skilled in the art. After transformation, positive clones wereselected, plasmid DNA was isolated and the sequence of the DKR-3 geneinsert was confirmed.

The construct pAMG21-human DKR-3-23-350 was engineered to be 329 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-Pro-Ala-Pro-Thr-Ala (SEQ ID NO:55) Gly-Gly-Glu-Glu-Ile. (SEQ IDNO:56)The template used for PCR was human DKR-3 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct.

(SEQ ID NO:57) GTTCTCCTCATATGCCTGCTCCAACTGCAACTTCGGCTCCAGTCAAGCCC GGCC(SEQ ID NO:58) TACGCACTCCGCGGTTAAATCTCTTCCCCTCCCAGCA

The construct pAMG21-human DKR-3-33-350 was engineered to be 319 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-Lys-Pro-Gly-Pro-Ala (SEQ ID NO:59) Gly-Gly-Glu-Glu-Ile (SEQ IDNO:60)The template used for PCR was human DKR-3 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct:

(SEQ ID NO:61) GTTCTCCTCATATGAAACCAGGTCCAGCCTTAAGCTACCCGCAGGAGGAG GCCA(SEQ ID NO:62) TACGCACTCCGCGGTTAAATCTCTTCCCCTCCCAGCA

The construct pAMG21-human DKR-3-42-350 was engineered to be 310 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-Gln-Glu-Glu-Ala-Thr (SEQ ID NO:63) Gly-Gly-Glu-Glu-Ile (SEQ IDNO:64)

The template used for PCR was human DKR-3 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct:

(SEQ ID NO:65) GTTCTCCTCATATGCAAGAAGAAGCTACTCTGAATGAGATGTTCCGCGAG GTT(SEQ ID NO:66) TACGCACTCCGCGGTTAAATCTCTTCCCCTCCCAGCA

The construct pAMG21-mouse DKR-3-33-349 was engineered to be 318 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-Glu-Pro-Gly-Pro-Ala (SEQ ID NO:67) Gly-Glu-Glu-Glu-Ile (SEQ IDNO:68)The template used for PCR was mouse DKR-3 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct:

(SEQ ID NO:69) GTTCTCCTCATATGGAACCAGGTCCAGCTTTAAACTACCCTCAGGAGGAA GCTA(SEQ ID NO:70) TACGCACTCCGCGGTTAAATCTCCTCCTCTCCGCCTA

Transformed E. coli GM121 containing the various pAMG21 DKR-3 plasmidsdescribed above were grown in 2× YT media containing 20 micrograms/mlkanamycin at 30° C. until the culture reached an optical density at 600nm of about 0.5. Induction of DKR-3 polypeptide expression was achievedby addition of Vibrio fischeri synthetic autoinducer to 100 ng/ml finalconcentration and incubation of the culture at either 30 or 37 C forabout 6 hours further with shaking. In addition, as a uninduced control,for each culture no autoinducer was added to an aliquot of the culture,but the culture was also incubated for about 6 hours further at 30 Cwith shaking along with the induced cultures. After about 6 hours, theoptical density of cultures were measured at about 600 nm, an aliquot ofcultures were examined by oil emersion microscopy at 1600×magnification, and aliquots of cultures were pelleted by centrifugation.Bacterial pellets of cultures were processed for SDS-polyacrylamide gelelectrophoresis to examine levels of protein produced in crude lysates,or bacterial pellets were processed to determine whether the recombinantprotein was in the soluble or insoluble fraction of E. coli and forN-terminal sequencing confirmation of the recombinant gene product. Theresults are shown as photos of the SDS gels in FIGS. 18 and 19. In FIG.18, Lane 10 is molecular weight markers, and Lanes 1-9 are crude lystesof bacterial cells. Lane 1 is crude lysate of uninduced control cells;Lanes 2, 4, 6, and 8 are crude lysates of induced cells cultured at 30C; Lanes 3, 5, 7, and 9 are induced cells cultured at 37 C. Lanes 1-5contain lysates of cells transfected with the pAMG21-human DKR-3-23-350construct; and Lanes 6-9 contain lysates of cells transfected with thepAMG21-human DKR-3-33-350 construct. The arrows to the left of Lane 2and the right of Lane 9 indicate the expected location of the DKR-3polypeptides. FIG. 19 contains molecular weight markers in Lane 10;Lanes 1-5 are crude lysates of cultured cells transfected with thepAMG21-human DKR-3-42-350 construct; Lanes 6-9 are crude lysates ofcells transfected with the pAMG21-mouse DKR-3-33-349 construct. Lanes 1and 6 are uninduced controls; Lanes 2, 4, 7, and 8 are crude lysates ofinduced cells cultured at 30 C (two different clones of each construct);Lanes 3, 5, and 9 are crude lysates of induced cells cultured at 37 C(two separate clones of the human DKR-3-42-350 construct in Lanes 3 and5). The arrow to the right of Lane 9 indicates the expected location ofthe mouse DKR-3 polypeptides; the arrow to the left of Lane 4 indicatesthe expected location of human DKR-3 polypeptide. As can be seen, allDKR-3 constructs produced large amounts of recombinant protein in E.coli. No inclusion bodies could be detected by oil emersion microscopy,and the recombinant polypeptides were mostly found in the solublefraction of the cells.

Example 10 Expression of DKR-4 in Bacteria

PCR amplification employing the primer pairs and template describedbelow were used to generate a recombinant form of human DKR-4. Oneprimer of each pair introduces a TAA stop codon and a unique BamHI sitefollowing the carboxy terminus of the gene. The other primer of eachpair introduces a unique NdeI site, a N-terminal methionine, andoptimized codons for the amino terminal portion of the gene. PCR andthermocycling was performed using standard recombinant DNA methodology.The PCR products were purified, restriction digested, and inserted intothe unique NdeI and BamHI sites of the vector pAMG21 (ATCC accession no.98113) and transformed into the prototrophic E. coli host GM94. Othercommonly used E. coli expression vectors and host cells are alsosuitable for expression. After transformation, positive clones wereselected and will be examined for expression of the recombinant geneproduct.

The construct pAMG21-human DKR-4-19-224 was engineered to be 207 aminoacids in length and have the following N-terminal and C-terminalresidues, respectively:

Met-Leu-Val-Leu-Asp-Phe (SEQ ID NO:71) Lys-Ile-Glu-Lys-Leu (SEQ IDNO:72)The template used for PCR was human DKR-4 cDNA and the followingoligonucleotides were the primer pair used for PCR and cloning this geneconstruct:

(SEQ ID NO:73) GTTCTCCTCATATGTTAGTTTTGGATTTCAACAACATCAGGAGCTCT (SEQ IDNO:74) TACGCACTGGATCCTTACAGTTTTTCTATTTTTTGGCATACTCTTAATC

It is anticipated that DKR-4 polypeptide could be prepared using the PCRproduct as described above for the other DKR polypeptides.

Example 11 Production and Purification of DKR-3 Polypeptide in MammalianCells

Human DKR-3 cDNA was cloned onto the mammalian expression vectorpcDNA3.1 (−)/mycHis (Invitrogen, Carlsbad, Calif.) and the vectorconstruct was amplified using the Qiagen maxi-prep kit (Qiagen,Chatsworth, Calif.) standard ligation techniques.

Human embryonic kidney 293T cells (American Type Culture Collection)were cultured in 10 cm dishes, and grown to about 80 percent confluence.The cells were then transfected with the vector construct using theDMRIE-C® liposome formulation (Gibco BRL, Grand Island, N.Y.) asfollows. About 240 microliters of DMRIE-C® were added to 8 ml of Optimemmedium. About 40 ul (equivalent to about 56 micrograms) of purifiedvector construct was then added to another 8 ml of Optimem. After mixingand incubation at room temperature for about 15 minutes, 2 ml of thissolution was added to each of 8 plates. After about 5 hours, the mediumwas aspirated and 10 ml of DME medium containing about 10 percent fetalcalf serum was added. The cells were incubated 16-18 hours after whichthe medium was removed and about 10 ml of SF Optimem medium per wellwithout phenol red were added. After about 24 hours, this medium, the“conditioned medium” was harvested, passed over a 0.22 micron filter andstored at 4° C. The cells were then incubated in another 10 ml of SFOptimem per plate. After 24 hours, this medium was collected, filteredand also stored at 4° C.

The conditioned media was added to a buffer containing 50 mM NaP0₄, pH8,and 250 mM sodium chloride, and passed over a column of nickel-Sephadex(Qiagen, Chatsworth, Calif.). Non-specifically bound proteins wereeluted using the same buffer containing 10 mM imidazole, followed by thesame buffer containing 20 mM imidazole. DKR-3 was then eluted using 125mM-250 mM imidazole. Fractions from the column were subjected to 12percent SDS gel electrophores and silver stained. The results are shownin FIG. 20. Lane 2 contains material that was loaded on to the gel. Lane3 contains the flow through fraction after loading the column withconditioned medium, Lanes 4, 5, 6, and 7 contain column fractions aftertreatment with 10, 20, 125, and 250 mM imidazole. Molecular weightstandards are shown in Lane 8. As can be seen a single band of proteinof the correct molecular weight is seen in Lanes 5 and 6, indicatingthat this procedure resulted in generation of purified DKR-3 protein(attached to myc and His tags). Smearing of the protein band may be dueto glycosylation. Separately, a Western blot was run to confirm that thepurified protein did indeed have a His tag (indicating that the fusionprotein DKR-3 mycHis had been produced). The Western blot was preparedusing standard procedures and was proved with a polyclonal anti-His-HRPantibody (Invitrogen, Carlsbad, Calif.). A photo of the Western blot isshown in FIG. 21; the Lanes correspond to that for the gel (describedimmediately above). As can be seen, there is antibody binding in Lanes2, 5, and 6, indicating that DKR-3 mycHis was loaded on to the columnand was eluted in the 20 and 125 mM imidazole washes.

Example 12 Anchorage Independent Growth Assay

A distinguishing feature of many cancer cell lines is their ability togrow in an anchorage independent manner. Whereas normal cells will onlygrow and divide until they come in contact with their neighbors, cancercells continue to grow and divide after contact, thereby forming tumors.Thus, one assay for cancer cell growth inhibitor compounds measures theability of cancer cells to grow and divide in the presence of thecompound. There are many ways known to the skilled artisan in which thisassay can be conducted, however two preferred methods are set forthbelow.

A. Stably Transfected Cell Assay

In this procedure, any human cancer cell line that does not express theDKR gene to be tested (either human DKR-1, 2, 3, 4, or a fragment orvariant thereof) is transfected with the DKR gene under evaluation,where the DKR gene is inserted into a vector such as pcDNA3.1(Invitrogen, Carlsbad, Calif.) or other suitable mammalian expressionvector. Transfection can be conducted as described herein. Thetransfected cancer cells are cultured to generate a stably transfectedcell line. Once a stably transfected cell line has been established, thecells are added to Noble or equivalent agar (about 0.35 percent)prepared in standard mammalian cell culture medium such as RPMI. Thecell/agar solution is poured on to petri plates containing solidifiedagar ban (about 0.5 percent agar). Colony formation is evaluated dailyto determine the rate of growth of the cells, and culture medium isadded to each plate as needed. Separately, the same cells aretransfected with vector only (containing no DKR gene). These “control”cells are then treated in an identical manner to the DKR gene containingcells and can be used as a standard of comparison for the DKR genecontaining cells.

Examples of suitable cancer cell lines for conducting this assayinclude, without limitation, human breast cancer cell line MCF7 and theglioblastoma cell line U-87MG.

B. Protein Assay

An alternate or additional assay to measure the growth of cancer celllines treated with a DKR polypeptide is as follows. Any human cancercell line not expressing the DKR polypeptide under evaluation can becultured and prepared with an agar solution as described above. Thecells can then be plated as described, and a solution of DKR polypeptide(either full length, or a fragment or variant thereof) in culture mediumcan be added to the agar either daily, every other day, or once per weekfor three weeks. Typically, a concentration of about 10 nM will beadded, although a series of dilutions ranging from 1 nM to 1 mM can beused. Control plates will receive a solution of culture medium only. Theplates can be monitored daily for up to about three weeks to evaluatecell colony formation. After three weeks, control and experimentalplates can be compared for the number and size of cell colonies. It isanticipated that those plates receiving DKR polypeptide that isbiologically active will have fewer cell colonies, and the colonies willbe smaller, as compared to control plates.

Example 13 In Vivo Tumor Assay

The ability of each DKR polypeptide to inhibit tumor growth in vivo canbe evaluated as follows. Tumor cells not expressing the DKR gene underevaluation can be transfected using procedures described herein with aDKR nucleic acid construct encoding a full length DKR gene, or afragment or variant thereof. The transfected cells can be maintained inculture (as described herein) until ready for use.

Male or female athymic nude mice (Charles River Labs, Boston, Mass.) arekept in a sterile environment. The mice are then injected with about2×10⁶ cells (either DKR transfected cells or control “vector only”transfected cells) in a total volume of about 0.1 ml can be injectedsubcutaneously. The mice can then be examined daily for appearance of(a) tumor(s) and for the size of the tumor. Preferably, the mice will beexamined for up to about six months so as to provide time for tumorgrowth (and regression where DKR polypeptides are effective atdecreasing tumor growth). The tumor(s), where present, can then beremoved, weighed and examined for (1) the presence of DKR polypeptide,and (2) morphology. Tumors from mice containing DKR constructtransfected cells can be compared to tumors from mice containing cellstransfected with vector only. It is anticipated that DKR polypeptides,due to their similarity with dkk-1, a potent wnt8 antagonist, will beable to decrease the size of the tumor as compared with controls.

1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO:13.
 2. The isolated polypeptide of claim 1, wherein the polypeptideis linked to a polymer.
 3. The isolated polypeptide of claim 2, whereinthe polymer is selected from the group consisting of a polyethyleneglycol polymer, dextran, cellulose and polyvinyl alcohol.